Identification of M-CSF agonists and antagonists

ABSTRACT

The present invention is directed to methods for crystallizing macrophage colony stimulating factor. The present invention is also directed to methods for designing and producing M-CSF agonists and antagonists using information derived from the crystallographic structure of M-CSF. The invention is also directed to methods for screening M-CSF agonists and antagonists. In addition, the present invention is directed to an isolated, purified, soluble and functional M-CSF receptor.

Work described herein was funded with Government support. The Governmenthas certain rights in inventions arising as part of that work.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. application Ser. No. 08/351,292 filedDec. 9, 1994; which is the U.S. national phase of internationalapplication PCT/US93/05548 filed on Jun. 9, 1993; and acontinuation-in-part of U.S. application Ser. No. 07/896,512 filed Jun.9, 1992 (now abandoned).

FIELD OF THE INVENTION

The present invention relates in general to crystalline compositions ofmacrophage colony stimulating factor "M-CSF" and in particular tomethods for the use of structural information (including X-raydiffraction patterns) of crystalline M-CSF for agonist and antagonistproduction, as well as assays for detection of same.

BACKGROUND OF THE INVENTION

Monocyte-macrophage colony-stimulating factor is produced by a varietyof cells, including macrophages, endothelial cells and fibroblasts (see,Ralph et al., "The Molecular and Biological Properties of the Human andMurine Members of the CSF-1 Family" in Molecular Basis of LymphokineAction, Humana Press, Inc., (1987), which is incorporated herein byreference). M-CSF is composed of two "monomer" polypeptides, which forma biologically active dimeric M-CSF protein (hereinafter referred to as"M-CSF dimer"). M-CSF belongs to a group of biological agonists thatpromote the production of blood cells. Specifically, it acts as a growthand differentiation factor for bone marrow progenitor cells of themononuclear phagocyte lineage. Further, M-CSF stimulates theproliferation and function of mature macrophages via specific receptorson responding cells. In clinical trials M-CSF has shown promise as apharmaceutical agent in the correction of blood cell deficienciesarising as a side-effect of chemotherapy or radiation therapy for cancerand may be beneficial in treating fungal infections associated with bonemarrow transplants. M-CSF may also play significant biological roles inpregnancy, uveitis, and atherosclerosis. Development of M-CSF agonistsor antagonists may prove to be of value in modifying the biologicalevents involved in these conditions.

M-CSF exists in at least three mature forms: short (M-CSFα),intermediate (M-CSF-γ), and long (M-CSFβ). Mature M-CSF is defined asincluding polypeptide sequences contained within secreted M-CSFfollowing amino terminus processing to remove leader sequences andcarboxyl terminus processing to remove domains including a putativetransmembrane region. The variations in the three mature forms are dueto alternative mRNA splicing (see, Cerretti et al. Molecular Immunology,25:761 (1988)). The three forms of M-CSF are translated from differentmRNA precursors, which encode polypeptide monomers of 256 to 554 aminoacids, having a 32 amino acid signal sequence at the amino terminal anda putative transmembrane region of approximately 23 amino acids near thecarboxyl terminal. The precursor peptides are subsequently processed byamino terminal and carboxyl terminal proteolytic cleavages to releasemature M-CSF. Residues 1-149 of all three mature forms of M-CSF areidentical and are believed to contain sequences essential for biologicalactivity of M-CSF. In vivo M-CSF monomers are dimerized viadisulfide-linkage and are glycosylated. Some, if not all, forms of M-CSFcan be recovered in membrane-associated form. Such membrane-bound M-CSFmay be cleaved to release secreted M-CSF. Membrane associated M-CSF isbelieved to have biological activity similar to M-CSF, but may haveother activities including cell-cell association or activation.

Polypeptides, including the M-CSFs, have a three-dimensional structuredetermined by the primary amino acid sequence and the environmentsurrounding the polypeptide. This three-dimensional structureestablishes the polypeptide's activity, stability, binding affinity,binding specificity, and other biochemical attributes. Thus, a knowledgeof a protein's three-dimensional structure can provide much guidance indesigning agents that mimic, inhibit, or improve its biological activityin soluble or membrane bound forms.

The three-dimensional structure of a polypeptide may be determined in anumber of ways. Many of the most precise methods employ X-raycrystallography (for a general review, see, Van Holde, PhysicalBiochemistry, Prentice-Hall, N.J. pp. 221-239, (1971), which isincorporated herein by reference). This technique relies on the abilityof crystalline lattices to diffract X-rays or other forms of radiation.Diffraction experiments suitable for determining the three-dimensionalstructure of macromolecules typically require high-quality crystals.Unfortunately, such crystals have been unavailable for M-CSF as well asmany other proteins of interest. Thus, high-quality, diffractingcrystals of M-CSF would assist the determination of itsthree-dimensional structure.

Various methods for preparing crystalline proteins and polypeptides areknown in the art (see, for example, McPherson, et al. "Preparation andAnalysis of Protein Crystals", A. McPherson, Robert E. KriegerPublishing Company, Malabar, Fla. (1989); Weber, Advances in ProteinChemistry 41:1-36 (1991); U.S. Pat. No. 4,672,108; and U.S. Pat. No.4,833,233; all of which are incorporated herein by reference for allpurposes). Although there are multiple approaches to crystallizingpolypeptides, no single set of conditions provides a reasonableexpectation of success, especially when the crystals must be suitablefor X-ray diffraction studies. Thus, in spite of significant research,many proteins remain uncrystallized.

In addition to providing structural information, crystallinepolypeptides provide other advantages. For example, the crystallizationprocess itself further purifies the polypeptide, and satisfies one ofthe classical criteria for homogeneity. In fact, crystallizationfrequently provides unparalleled purification quality, removingimpurities that are not removed by other purification methods such asHPLC, dialysis, conventional column chromatography, etc. Moreover,crystalline polypeptides are often stable at ambient temperatures andfree of protease contamination and other degradation associated withsolution storage. Crystalline polypeptides may also be useful aspharmaceutical preparations. Finally, crystallization techniques ingeneral are largely free of problems such as denaturation associatedwith other stabilization methods (e.g. lyophilization). Thus, thereexists a significant need for preparing M-CSF compositions incrystalline form and determining their three-dimensional structure. Thepresent invention fulfills this and other needs. Once crystallizationhas been accomplished, crystallographic data provides useful structuralinformation which may assist the design of peptides that may serve asagonists or antagonists. In addition, the crystal structure providesinformation useful to map, the receptor binding domain which could thenbe mimicked by a small non-peptide molecule which may serve as anantagonist or agonist.

SUMMARY OF THE INVENTION

The present invention provides crystalline forms of M-CSF dimers.Preferably, the dimers are formed from polypeptides containing between146 to 162 amino acids residues at or near the N-terminus of matureM-CSF (e.g. glu₁ glu₂ val₃ . . .). In a specific embodiment, thepolypeptide includes residues 4 to 158 of mature M-CSFα polypeptide,preferably in the non-glycosylated form.

Another aspect of the invention provides a method of crystallizing anM-CSF. A preferred crystallization method according to the presentinvention includes the following steps: mixing a preselected,substantially pure M-CSF dimer and a precipitant to form an M-CSFmixture; precipitating crystalline M-CSF from the mixture; and isolatingthe M-CSF in crystalline form. In some specific embodiments, theprecipitant contains polyethylene glycol. Other components such asammonium sulfate and/or other ionic compounds may be added to thesolution. It has been found by x-ray crystallography that M-CSF producedby the method of the present invention can crystallize into the P2₁ 2₁2₁ space groups for example.

Variations of the crystallization method are also provided. For example,the step of precipitating crystals from the M-CSF mixture may involveequilibrating the M-CSF mixture with a second mixture. The secondmixture is typically a solution that consists of a higher concentrationof precipitant than the first M-CSF mixture. The step of equilibratingpreferably consists of applying the M-CSF mixture to a surface andallowing the applied M-CSF mixture to come into equilibrium with areservoir of the second mixture. In other embodiments, the step ofprecipitating M-CSF crystals is initiated by seeding the M-CSF mixturewith seed crystals or altering the temperature of the M-CSF mixture.Another aspect of the invention involves identifying compounds that havestructures that mimic a receptor binding region of the three-dimensionalstructure of M-CSF to varying degrees and can in many instances functionas M-CSF agonists or antagonists. Compounds that interact with thereceptor-binding region of M-CSF may be antagonists. Thethree-dimensional alpha-carbon coordinates of a truncated M-CSF dimer ispresented in Appendix 1. In one embodiment of the present invention, thethree-dimensional structure of M-CSF is obtained by first crystallizingan M-CSF dimer (having M-CSF receptor-binding residues) to form at leastone M-CSF crystal. Next, a source of radiation is used for irradiatingan M-CSF crystal to obtain a diffraction pattern of the M-CSF crystal.Finally, a three-dimensional structure of M-CSF is obtained from thediffraction pattern. In most embodiments, the three-dimensionalstructure includes an M-CSF receptor-binding region.

The present invention is also directed to a method for selectingcandidate amino acid substitutions in a protein, based on structuralinformation, and more particularly M-CSF, comprising determining thethree-dimensional structure of M-CSF by the methods of the presentinvention; followed by determining the solvent accessible amino acidresidues of the protein, determining which residues are not involved indimer formation. Applying these criteria, amino acids in M-CSF which aresolvent accessible and which are not involved in dimer formation areselected for substitution with non-conservative amino acids. SinceM-CSFβ has intrachain disulfide bonds involving cysteines 157 and/or159, we believe the C-terminal region of M-CSF extends from the "rear"of the structure we have solved, providing a variable-length "tether"for membrane-bound forms of M-CSF. Thus, the "front" or receptor-bindingregion of M-CSF is on the opposite side of the molecules, consisting ofsolvent-accessible residues in or near helices A, C, and D, includingresidues from about 6 to 26, 71 to 90, and 110 to 130, respectively, ofnative M-CSF. Preferred amino acids for substitution and preferredsubstituting amino acids include but are not limited to: H15→A or L;Q79→A or D; R86→E or D; E115→A; E41→K or R; K93→A or E; D99→K or R;L55→Q or N; S18→A or K; Q20→A or D; I75→K or E; V78→K or R; L85→E or N;D69→K or R; N70→A or E; H9→A or D; N63→K or R; and T34→Q or K. Mostpreferred are those substitutions give rise to novel M-CSF agonists andM-CSF antagonists. Additionally, the present invention is also directedto a method for producing antagonists and agonists by substituting atleast one and preferably fewer than 5 solvent accessible residues perM-CSF monomer.

The invention is also directed to heterodimeric M-CSF in which only onesubunit contains substituted solvent accessible amino acids involved insignal transduction and to heterodimeric M-CSF in which each subunitcontains different substituted solvent accessible amino acids involvedin signal transduction. The present invention is also directed to M-CSFhaving amino acid substitutions which do not impair binding to the M-CSFreceptor. Screening for agonists and antagonists is then accomplishedusing bioassays and receptor binding assays using methods well known inthe art, including those described in the Examples below.

In addition the invention is directed to an isolated, purified, solubleand functional M-CSF receptor. The present invention is also directed toa method for screening M-CSF agonists and antagonists using a solubleM-CSF receptor.

A further understanding of the present invention can be obtained byreference to the drawings and discussion of specific embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a section of a diffraction pattern of an M-CSFα crystalprepared according to the present invention;

FIG. 2 is a topology diagram showing the disulfide bonds in truncateddimeric M-CSF;

FIG. 3 is a stereodiagram of the C-alpha backbone with every tenthresidue labelled and with the non-crystallographic symmetry axisindicated by a dotted line;

FIGS. 4 and 4A present two views of a ribbon diagram highlighting thesecondary structural elements of M-CSF. The cysteine residues have beenrepresented by a ball-and-stick model and the non-crystallographicsymmetry axis is indicated by a dotted line;

FIGS. 5A-5C illustrate size-exclusion HPLC analysis of the NΔ3CΔ158M-CSF short clone homodimer (158), NΔ3CΔ221 C157S, C159S long clonehomodimer (221F), and the short clone/long clone heterodimer (158/221F)and their corresponding biological activities;

FIGS. 6A-6C illustrate size exclusion HPLC and both non-reduced andreduced SDS-PAGE analysis of the preparative purification of M-CSF moreparticularly, FIG. 6A graphically illustrates the separation onPhenyl-HPLC size exclusion chromatography of the three species of M-CSFdimers of FIGS. 5A-5C, i.e., the 158 homodimer, the 221F homodimer andthe 158/221F heterodimer, and indicates that the three absorbance peaksat 280 nm (solid line) correlate with M-CSF activity in U/ml×10⁻⁶(dotted line); FIG. 6B illustrates an SDS-PAGE analysis undernon-reducing conditions of the preparative purification of the 158/221Fheterodimer (intermediate molecular weight species) relative to the 158homodimer (lower molecular weight species) and the 221F homodimer(highest molecular weight species); FIG. 6C illustrates an SDS-PAGEanalysis under reducing conditions of the preparative purification ofthe 158/221F heterodimer (middle lanes) relative to the 158 homodimer(left lanes) and the 221F homodimer (right lanes); and

FIG. 7 illustrates the competitive binding of M-CSF and M-CSF muteins toNFS60 cell M-CSF receptors. In FIG. 7, competitive binding curves areshown for M-CSFα NΔ3CΔ158 (closed circles); M-CSFα NΔ3CΔ158 H9A,H15A/M-CSFβ NΔ3CΔ221 C157S, C159S heterodimer (closed squares); dimericQ20A, V78KF mutein (open circles); and dimeric H9A, H15A mutein (opensquares).

DESCRIPTION OF THE PREFERRED EMBODIMENTS DEFINITIONS

As used herein "M-CSF polypeptide" refers to a human polypeptide havingsubstantially the same amino acid sequence as the mature human M-CSFα,M-CSFβ, or M-CSFγ polypeptides described in Kawasaki et al., Science230:291 (1985), Cerretti et al., Molecular Immunology, 25:761 (1988), orLadner et al., EMBO Journal 6:2693 (1987), each of which areincorporated herein by reference. Such terminology reflects theunderstanding that the three mature M-CSFs have different amino acidsequences, as described above.

Certain modifications to the primary sequence of M-CSF can be made bydeletion, addition, or alteration of the amino acids encoded by the DNAsequence without destroying the desired structure (e.g., the receptorbinding ability of M-CSF) in accordance with well-known recombinant DNAtechniques. Further, a skilled artisan will appreciate that individualamino acids may be substituted or modified by oxidation, reduction orother derivitization, and the polypeptide may be cleaved to obtainfragments that retain the active binding site and structuralinformation. Such substitutions and alterations result in polypeptideshaving an amino acid sequence which falls within the definition ofpolypeptide "having substantially the same amino acid sequence as themature M-CSFα, M-CSFβ, and M-CSFγ polypeptides."

For purposes of crystallization, preferred lengths of the M-CSFα, β or γmonomers are between about 145 and 180 amino acids (counting from themature amino terminus), and more preferably between about 145 and 162amino acids long. A specific monomer that may be present in acrystallizable dimer is M-CSFα and is NΔ3 M-CSFα CΔ158 (3 amino acidsare deleted from the amino terminus and the total length is 155 aminoacids). All lengths are inclusive. As used herein the term "M-CSFα(4-158)" denotes an M-CSF having amino acid residues 4 to 158 of themature, processed M-CSFα polypeptide. Other nomenclature designationsfor C-terminal and N-terminal truncations of native M-CSF are set forthin U.S. Pat. No. 4,929,700 which is incorporated herein by reference.

Crystallizable glycosylation variants of the M-CSF polypeptides areincluded within the scope of this invention. These variants includepolypeptides completely lacking in glycosylation and variants having atleast one fewer glycosylated site than the mature forms, as well asvariants in which the glycosylation pattern has been changed from thenative forms. Also included are deglycosylated and unglycosylated aminoacid sequence variants, as well as deglycosylated and unglycosylatedM-CSF subunits having the mature amino acid sequence (see, U.S. Pat. No.5,032,626).

"M-CSF" dimer refers to two M-CSF polypeptide monomers that havedimerized. M-CSF dimers may include two identical polypeptide monomers(homodimers) or two different polypeptide monomers (heterodimers such asan M-CSFα-M-CSFβ dimer, an M-CSF long clone and short clone dimer).M-CSF monomers may be converted to M-CSF dimers in vitro as described inU.S. Pat. No. 4,929,700, which is incorporated herein by reference.Recombinantly expressed M-CSFs may also be variably glycosylated as theyexist in vivo, partially glycosylated, or completely lacking inglycosylation (unglycosylated). Glycosylated M-CSFs may be produced invivo with carbohydrate chains which may later be enzymaticallydeglycosylated in vitro.

Biologically active M-CSF exhibits a spectrum of activity understood inthe art. For instance, M-CSF stimulates the proliferation and functionof mature macrophages via specific receptors on responding cells.Further, M-CSF acts as a mononuclear phagocyte progenitor growth factor.The standard in vitro colony stimulating assay of Metcalf, J. CellPhysiol. 76:89 (1970) (which is incorporated herein by reference)results primarily in the formation of macrophage colonies when M-CSF isapplied to stem cells. Other biological assays are based on M-CSFinduced proliferation of M-CSF dependent cells such as the NFS-60 cellline. As used herein "M-CSF having biological activity" refers to M-CSF,including fragments and sequence variants thereof as described herein;that exhibit an art-recognized spectrum of activity with respect tobiological systems. Such M-CSF having biological activity will typicallyhave certain structural attributes in common with those of the matureM-CSF forms such as receptor binding site tertiary structure.

Agonists are substances that exhibit greater activity per se than thenative ligand while antagonists are substances that suppress, inhibit,or interfere with the biological activity of a native ligand. Agonistsand antagonists may be produced by the methods of the present inventionfor use in the treatment of diseases in which M-CSF has been implicatedeither as a potential treatment (e.g., for treating blood celldeficiencies arising as a side effect of chemotherapy treating fungalinfection associated with bone marrow transplants and others) or ashaving a role in the pathogenesis of the disease (e.g., ovarian cancer,uveitis, atherosclerosis).

Crystallization of M-CSF species in accordance with the presentinvention includes four general steps: expression, purification,crystallization and isolation.

Expression of Recombinant M-CSF

M-CSF crystallization requires an abundant source of M-CSF that may beisolated in a relatively homogeneous form. A variety of expressionsystems and hosts are suitable for the expression of M-CSF and will bereadily apparent to one of skill in the art. Because of the variabilityof glycosylation and other post-transnational modifications present inM-CSF produced in certain eukaryotic hosts, expression in E. coli mayprovide M-CSF with advantageous properties with regard tocrystallization. Typical in vitro M-CSF expression systems are describedin U.S. Pat. No. 4,929,700, for example.

For use in the present invention, a variety of M-CSF polypeptides canalso be readily designed and manufactured utilizing recombinant DNAtechniques well known to those skilled in the art. For example, theM-CSF amino acid sequence can vary from the naturally occurring sequenceat the primary structure level by amino acid substitutions, insertions,deletions, and the like. These modifications can be used in a number ofcombinations to produce the final modified polypeptide chain. Thepresent invention is useful for crystallizing such polypeptides anddimers thereof.

In general, modifications of the genes encoding the M-CSF polypeptideare readily accomplished by a variety of well-known techniques, such assite-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979)and Roberts, S. et al., Nature 328:731-734 (1987) and U.S. Pat. No.5,032,676, all of which are incorporated herein by reference). Mostmodifications are evaluated by screening in a suitable assay for thedesired characteristic. For instance, a change in the M-CSFreceptor-binding character of the polypeptide can be detected bycompetitive assays with an appropriate reference polypeptides or by thebioassays described in U.S. Pat. No. 4,847,201, which is incorporatedherein by reference.

Insertional variants of the present invention are those in which one ormore amino acid residues are introduced into a predetermined site in theM-CSF. For instance, insertional variants can be fusions of heterologousproteins or polypeptides to the amino or carboxyl terminus of thesubunits. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Nonnatural amino acids (i.e., amino acids not normally found in nativeproteins), as well as isosteric analogs (amino acid or otherwise) arealso suitable for use in this invention. Examples of suitablesubstitutions are well known in the art, such as the Glu->Asp, Ser->Cys,and Cys->Ser, His->alanine for example. Another class of variants aredeletional variants, which are characterized by the removal of one ormore amino acid residues from the M-CSF.

Other variants of the present invention may be produced by chemicallymodifying amino acids of the native protein (e.g., diethylpyrocarbonatetreatment which modifies histidine residues). Preferred or chemicalmodifications which are specific for certain amino acid side chains.Specificity may also be achieved by blocking other side chains withantibodies directed to the side chains to be protected. Chemicalmodification includes such reactions as oxidation, reduction, amidation,deamidation, or substitution of bulky groups such as polysaccharides orpolyethylene glycol (see e.g., U.S. Pat. No. 4,179,337 and WO91/21029both of which are incorporated herein by reference).

Exemplary modifications include the modification of lysinyl and aminoterminal residues by reaction with succinic or other carboxylic acidanhydrides. Modification with these agents has the effect of reversingthe charge of the lysinyl residues. Other suitable reagents formodifying amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea, 2,4-pentanedione; andtransaminase-catalyzed reaction with glyoxylate, andN-hydroxysuccinamide esters of polyethylenene glycol or other bulkysubstitutions.

Arginyl residues may be modified by reaction with a number of reagents,including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, andninhydrin. Modification of arginine residues requires that the reactionbe performed in alkaline conditions because of the high pK_(a) of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

Tyrosyl residues may also be modified with particular interest inintroducing spectral labels into tyrosyl residues by reaction witharomatic diazonium compounds or tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues may also be iodinated using ¹²⁵ I or ¹³¹ I to prepare labeledproteins for use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R--N═C═N--R¹), where R and R¹ aredifferent alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Conversely, glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues, respectively, under mildlyacidic conditions. Either form of these residues falls within the scopeof this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

The availability of a DNA sequence encoding M-CSF permits the use ofvarious expression systems to produce the desired polypeptides.Construction of expression vectors and recombinant production from theappropriate DNA sequences are performed by methods well known in theart. These techniques and various other techniques are generallyperformed according to Sambrook et al., Molecular Cloning--A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989),and Kriegler, M., Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York (1990), both of which are incorporated hereinby reference.

Purification of M-CSF

Purification steps are employed to ensure that the M-CSF is isolated,prior to crystallization, in a relatively homogeneous state. In general,a higher purity solution increases the likelihood of success ofsubsequent crystallization steps. Typical purification methods includethe use of centrifugation, partial fractionation using salt or organiccompounds, dialysis, conventional column chromatography (such asion-exchange, molecular sizing chromatography etc.), high performanceliquid chromatography (HPLC), and gel electrophoresis methods (see,e.g., Deutcher, "Guide to Protein Purification" in Methods in Enzymology(1990), Academic Press, Berkely, Calif., which is incorporated herein byreference for all purposes). Preferred purification conditions forgenerating unusually homogeneous M-CSF species as well as purificationof these species are disclosed, for example, in U.S. Pat. No. 4,929,700which is incorporated herein by reference. Other purification methodsare known and will be apparent to one of skill in the art.

Crystallization of M-CSF

Although many of the same physical principles govern crystallization ofpolypeptides (including M-CSF dimers) and small molecules, the actualcrystallization mechanisms differ significantly. For example, thelattice of small-molecule crystals effectively excludes solvent whilethat of polypeptide crystals includes substantial numbers of solventmolecules. Thus, special techniques must typically be applied tocrystallize polypeptides.

Polypeptide crystallization occurs in solutions where the polypeptideconcentration exceeds its solubility maximum (i.e., the polypeptidesolution is supersaturated). Such "thermodynamically metastable"solutions may be restored to equilibrium by reducing the polypeptideconcentration, preferably through precipitation of the polypeptidecrystals. Often polypeptides may be induced to crystallize fromsupersaturated solutions by adding agents that alter the polypeptidesurface charges or perturb the interactions between the polypeptide andbulk water to promote associations that lead to crystallization.

Compounds known as "precipitants" are often used to decrease thesolubility of the polypeptide in a concentrated solution. Precipitantsinduce crystallization by forming an energetically unfavorableprecipitant depleted layer around the polypeptide molecules. To minimizethe relative amount of this depletion layer, the polypeptides formassociations and ultimately crystals as explained in Weber, Advances inProtein Chemistry 41:1-36 (1991) which was previously incorporated byreference. In addition to precipitants, other materials are sometimesadded to the polypeptide crystallization solution. These include buffersto adjust the pH of the solution (and hence surface charge on thepeptide) and salts to reduce the solubility of the polypeptide. Variousprecipitants are known in the art and include the following: ethanol,3-ethyl 1-2,4-pentanediol; and many of the polyglycols, such aspolyethylene glycol. A suitable precipitant for crystallizing M-CSF ispolyethylene glycol (PEG), which combines some of the characteristics ofthe salts and other organic precipitants (see, for example, Ward et al.,J. Mol. Biol. 98:161 [1975] which is incorporated herein by referencefor all purposes and McPherson J. Biol. Chem. 251:6300 [1976], which waspreviously incorporated by reference).

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchmethod, polypeptide is mixed with precipitants to achievesupersaturation, the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane which is placed into a solution containingprecipitant. Equilibration across the membrane increases the polypeptideand precipitant concentrations thereby causing the polypeptide to reachsupersaturation levels.

In the hanging drop technique, an initial polypeptide mixture is createdby adding a precipitant to concentrated polypeptide solution. Theconcentrations of the polypeptide and precipitants are such that in thisinitial form, the polypeptide does not crystallize. A small drop of thismixture is placed on a glass slide which is inverted and suspended overa reservoir of a second solution. The system is then sealed. Typicallythe second solution contains a higher concentration of precipitant orother dehydrating agent. The difference in the precipitantconcentrations causes the protein solution to have a higher vaporpressure than the solution. Since the system containing the twosolutions is sealed, an equilibrium is established, and water from thepolypeptide mixture transfers to the second solution. This equilibrationincreases the polypeptide and precipitant concentration in thepolypeptide solution. At the critical concentration of polypeptide andprecipitant, a crystal of the polypeptide will form. The hanging dropmethod is well known in the art (see, McPherson J. Biol. Chem. 251:6300[1976], which was previously incorporated herein by reference).

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

In preferred embodiments, the crystals of the present invention will beformed from a dimer of M-CSF polypeptides. Preferred crystals aretypically at least about 0.2×0.2×0.05 mm, more preferably larger than0.4×0.4×0.4 mm, and most preferably larger than 0.5×0.5×0.5 mm. Aftercrystallization, the protein may be separated from the crystallizationmixture by standard techniques.

The crystals so produced have a wide range of uses. For example, highquality crystals are suitable for X-ray or neutron diffraction analysisto determine the three-dimensional structure of the M-CSF and, inparticular, to assist in the identification of its receptor bindingsite. Knowledge of the binding site region and solvent-accessibleresidues available for contact with the M-CSF receptor allows rationaldesign and construction of agonists and antagonist for M-CSFs.Crystallization and structural determination of M-CSF muteins havingaltered receptor binding ability or bioactivity allows the evaluation ofwhether such changes are caused by general structural deformation or byside chain alteration at the substitution site.

In addition, crystallization itself can be used as purification method.In some instances, a polypeptide or protein will crystallize from aheterogeneous mixture into crystals. Isolation of such crystals byfiltration, centrifugation, etc. followed by redissolving thepolypeptide affords a purified solution suitable for use in growing thehigh-quality crystals necessary for diffraction studies. Thesehigh-quality crystals may also be dissolved in water and then formulatedto provide an aqueous M-CSF solution having various uses known in theart including pharmaceutical purposes.

Of course, amino acid sequence variants of M-CSF may also becrystallized and used. These mutants can be used for, among otherpurposes, obtaining structural information useful for directingmodification of the binding affinity for M-CSF receptors. As with thenaturally occurring forms, the modified M-CSF forms may be useful aspharmaceutical agents for stimulating bone marrow proliferation,overcoming immune suppression and fungal diseases induced bychemotherapy, improving therapeutic efficacy, and lessening the severityor occurrence of side effects during therapeutic use of the presentinvention. Furthermore, modified M-CSFs may be useful for treatment ofdisease in which soluble or membrane-bound M-CSF causes or exacerbatesthe disease state.

Characterization of M-CSF

After purification, crystallization and isolation, the subject crystalsmay be analyzed by techniques known in the art. Typical analysis yieldstructural, physical, and mechanistic information about the peptides. Asdiscussed above, X-ray crystallography provides detailed structuralinformation which may be used in conjunction with widely availablemolecular modeling programs to arrive at the three-dimensionalarrangement of atoms in the peptide. Exemplary modeling programs include"Homology" by Biosym (San Diego, Calif.), "Biograf" by BioDesign,"Nemesis" by Oxford Molecular, "SYBYL" and "Composer" by TriposAssociates, "CHARM" by Polygen (Waltham, Mass.), "AMBER" by Universityof California, San Francisco, and "MM2" and "MMP2" by Molecular Design,Ltd.

Peptide modeling can be used to design a variety of agents capable ofmodifying the activity of the subject peptide. For example, using thethree-dimensional structure of the active site, agonists and antagonistshaving complementary structures can be designed to enhance thetherapeutic utility of M-CSF treatment or to block the biologicalactivity of M-CSF. Further, M-CSF structural information is useful fordirecting design of proteinaceous or non-proteinaceous M-CSF agonistsand antagonists, based on knowledge of the contact residues between theM-CSF ligand and its receptor. Such residues are identified by the M-CSFcrystal structure as those which are solvent-accessible, distal to thecarboxyl terminal membrane anchoring region not involved in dimerinterface stabilizations, and possibly including residues not conservedbetween human and mouse M-CSF (which does not recognize the human M-CSFreceptor).

EXAMPLE 1

Systematic crystallization trials with M-CSF were made using the hangingdrop technique. A microdroplet (5μl) of mother liquor is suspended fromthe underside of a microscope cover slip, which is placed over a wellcontaining 1 ml of the precipitating solution. 60-70 initial trials wereset up, in which pH, temperature, counterion and precipitant werevaried. From these trials, the few that gave promising microcrystalswere picked for more careful examination.

It was discovered that suitable crystals may be grown from a 20 μl dropcontaining: 10 mg/ml protein, 100 mM MgCl₂, 50 mM Tris.Cl, pH 8.5, and12% PEG 4000. This drop was equilibrated against a reservoir containing24% PEG 4000. Tiny, needle-like crystals appeared in 2-3 days which wereredissolved in 10 μl water and recrystallized at room temperature. Goodquality chunky crystals appeared in 7-9 days in sizes ranging from0.3×0.3×0.3 mm to 0.5×0.5×1.0 mm.

Precession photographs revealed the space group to be P2₁ 2₁ 2₁ withunit cell dimensions: a=33.53 Å, b=65.11 Å, c=159.77 Å. This gives aunit cell volume of 349084.5 Å³, which is consistent with a dimer in thecrystallographic asymmetric unit, and 52% of the unit cell volume beingoccupied by solvent. The crystals diffracted to a resolution of 3 Å on aRigaku rotating anode X-ray generator (Danvers, Mass.) operated at 50 kVand 60 mA, and to 2.6 Å in synchrotron radiation.

Screening for heavy atom derivatives was done by soaking crystals intosolutions of heavy-metal salts. Zero-level precession pictures of thesoaks were used to identify potential derivatives. About 30 differentsoaking conditions were examined, of which 4 potential derivatives wereidentified. Unfortunately, some soaks caused the crystals to exhibitnon-isomorphism (i.e., the heavy atom soaks induced a change in celldimensions, making them unusable for phase calculation).

Three-dimensional intensity data were collected on film using anoscillation camera on the X-ray beam-line at the National SynchrotronLight Source, Brookhaven. Several other data sets, of native(underivatized M-CSF) as well as potential derivative crystals have beencollected on a Rigaku X-ray generator. The following data sets werecollected.

    ______________________________________                                                  Resolution                                                                             N          N                                                 Crystal (Å) (observations) (unique) X-Ray Source                        ______________________________________                                        Native    2.8      27922      7311  Synchrontron                                Native 2.9 35236 7002 Rigaku                                                      (film)                                                                    Native 3.5 5144 5116 Rigaku                                                       (diffractometer)                                                          K.sub.2 Hg(SCN).sub.4 3.5 15885 4119 Rigaku                                       (film)                                                                    UO.sub.2 Cl.sub.2 3.5 25492 5048 Rigaku                                           (film)                                                                    Cis-Pd(NH.sub.3).sub.2 Cl.sub.2 3.1 26122 6304 Synchrotron                  ______________________________________                                    

EXAMPLE 2

Recombinant M-CSF polypeptides were purified from E. coli. and renaturedto form a disulfide-linked dimeric protein as described in U.S. Pat. No.4,929,700. Crystallization of the resulting unglycosylated M-CSFαprotein (amino acids 4-158 in homodimeric form) was performed by thehanging drop method. Glass microscope plates were siliconized prior touse by dipping immersion into a 1% (volume:volume) solution of theorganosilane compound, Prosil-28 (PCR Incorporated, Gainesville, Fla.,32602) washing the treated glass plates with water, and baking at 180degrees.

A 2 mg/ml aqueous solution of purified human recombinant M-CSF wasdialyzed and concentrated against 50 mM Tris-HCl (pH 8.5) using adialysis tubing having a 10 kD cutoff. The final concentration ofpolypeptide (10 mg/ml) was determined by ultraviolet spectrophotometryat 280 nm.

About 7 microliters of the concentrated solution was mixed in each wellof the spot plate with 7 microliter of 20% (v/v) PEG 4000, 0.2 M MgCl₂,0.1 M Tris-HCl (pH 8.5). The spot plate was then placed in a clearplastic sandwich box containing 20 ml of 23% PEG 4000, 0.2 M MgCl₂, 0.1M Tris-HCl (pH 8.5) and the box was immediately sealed and stored atroom temperature. Minor variations in this procedure such as alteringbuffer conditions are within the scope of the present invention. Forexample, in a preferred embodiment of the present invention, bufferconditions were altered to include 150 mM MgCl₂ and 24% PEG 4000.

After 3-5 days, small microcrystals having a size of 0.1×0.1×0.05 mmappeared in each well. These microcrystals were isolated and redissolvedin 25 microliter of 50 mM Tris-HCl and allowed to stand at roomtemperature. The purified M-CSF crystallized from solution into largehexagonal prism shaped crystals ranging in size from 0.3×0.3×0.3 mm to 1mm×2 mm×0.5 mm. These crystals were stable at room temperature for atleast three months. In some instances, an artificial mother liquor wasprepared using 23% PEG 4000 and 150 mM MgCl₂ crystals were then added tothis mother liquor. In these cases, the crystals were removed from themother liquor immediately prior to analysis.

Using reducing and non-reducing SDS-PAGE analysis, the M-CSF in thecrystals was shown to be identical in molecular weight to thebiologically active starting material. Thus, the M-CSF structureobtained from the crystals is likely to be essentially identical to thestructure of biologically active M-CSF.

EXAMPLE 3

Glass microscope slides were prepared as described in Example 2. 7microliters of the same concentrated M-CSFα protein solution was mixedin each well of the spot plate with 7 microliter of 30% (v/v) PEG 4000,0.2 M ammonium acetate, 0.1 M acetate buffer (pH 7.5). The spot platewas then placed in a clear plastic sandwich box containing 20 ml of 30%PEG 4000, 0.2 M ammonium acetate, 0.1 M acetate buffer (pH 7.5) and thebox was immediately sealed and stored at room temperature. After 3-5days, thin, plate-like, fragile crystals having a size of approximately0.3×0.3×0.05 mm appeared.

EXAMPLE 4 Preliminary X-ray Analysis

X-ray crystallographic analysis using precession photographs showed thatthe crystals produced in Example 2 have an orthorhombic crystal latticein the P2₁ 2₁ 2₁ space group with cell dimensions a=33.54, b=65.26,c=159.63 d=90.0, c=90.0 and f=90.0 angstroms and diffract to a nominalresolution of 2.6 angstroms using synchrotron radiation. These dataprovided a unit cell volume of 348084.5 angstroms³, which is consistentwith a dimer in the crystallographic asymmetric unit with 52% of theunit cell volume being occupied by solvent. FIG. 1 is a 12-degreeprecession photograph of the Okl-zone section of the M-CSF crystal. Thephotograph was taken using a precession camera manufactured byEnraf-Nonius Company (Delft, Holland), mounted on a Rigaku RU-200 X-raygenerator operated at 50 kV and 50 mA.

EXAMPLE 5 Testing of M-CSF Receptor Binding Ability Using Soluble M-CSFReceptor

An essential step in the biological function of M-CSF in vivo is thebinding to the M-CSF receptor, also referred to as the c-fms geneproduct. Recombinant human soluble M-CSF receptor (rhsM-CSFR),representing amino acids 20 to 511 (Coussens, L et al., Nature, 320:277(1986)) was used as an in vitro assay reagent to test thereceptor-binding ability of M-CSF proteins. To generate a soluble formof the transmembrane receptor, only the extracellular domain of thehuman M-CSF receptor was expressed in a baculovirus/insect cellrecombinant expression system. In order to purify the soluble receptorwithout adversely effecting tertiary or quaternary structure,non-denaturing chromatographic methods were chosen, as described below.Other choices exist for the purification of the recombinant receptor.Affinity chromatography may be employed when either a suitable antibodyto or ligand for the receptor are available. Alternatively, "tags" maybe added to the C-terminus of the recombinant receptor, i.e., KT3antibody recognition sequence, and purified by an anti-tag antibody,i.e., KT3, column, for use in affinity chromatography. In expressionsystems in which the rhsM-CSFR is glycosylated, lectin chromatographycan be used to enrich for specific glycoproteins.

The rhsM-CSFR can be used to study ligand/receptor interactions as wellas ligand-induced receptor dimerization. The assay used to detectligand/receptor binding employed the use of size exclusion-HPLC,essentially as described in European Patent Application WO92/21029, C.Cunningham, et al., with the following modifications: the column usedwas a Superose 6 (Pharmacia LKB Biotechnology, Inc.) and the mobilephase was PBS at 0.5 ml/min and a M-CSF to rhsM-CSFR ratio of 1:2. Atthis ratio, the M-CSF/rhsM-CSFR complex chromatographed with an apparenthydrodynamic radius of 190,00 molecular weight, the molecular weightexpected for a M-CSF(rhsM-CSFR)₂ complex. Other assays may be employedto measure ligand/receptor binding or receptor dimerization such aschemical crosslinking and SDS-PAGE or immunoprecipitation and SDS-PAGE.Molecules that inhibit receptor dimerization but not ligand bindingprovide another method to antagonize M-CSF actions.

The DNA encoding rhsM-CSFR was cloned for expression in insect cellsusing the following general strategy. The portion of the c-fms genecorresponding to amino acids one to 511 was amplified from humanmacrophage cDNA by polymerase chain reaction (PCR) using an upstreamprimer of: 5'-GCGTACCATGGGCCCAGGAGTTCTGC-3' (SEQ ID NO.9) and adownstream primer of: 5'-AGTCGAGGATCCTCAATCCGGGGGATGCGTGTG-3' (SEQ IDNO.10). The underlined sequences are the NcoI and BamHI restrictionsites used to subclone the PCR product into the pAcC5 vector (Luckov etal., Bio/Technology 6:47-55). The pAcC5:hsM-CSFR vector was expressed inSF9 insect cells using a baculovirus helper vector as previouslydescribed (Summers, et al., A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures (1987)).

Approximately two liters of serum-free 72-hour conditioned medium wascollected by centrifugation and filtration from SF9 cells infected withpAcC5:hsM-CSFR construct described above. The material was diafilteredwith DEAE buffer A [10 mM Tris, pH 8.8, containing the followingprotease inhibitors (which were added all buffers throughout thepurification): 1 mM EDTA, 2 μg/ml leupeptin and 100 μM PMSF] andconcentrated 20-fold with a 20,000 molecular weight cutt-off PyrostatUltrafiltration Membrane (Sartorius). The retentate was loaded onto aDEAE Sepharose column (Pharmacia LKB Biotechnology, Inc., Piscataway,N.J.) having a bed volume of 100 ml that had been pre-equilibrated withDEAE Buffer A. Elution was at 5 ml/min with a 0-0.8 M NaCl gradient in500 ml of DEAE Buffer A. Fractions enriched in rhsM-CSFR were detectedWestern Analysis [Burnett, R., Anal. Biochem., 112:195 (1981)] and dotblot analysis of serially diluted fractions, using anti-c-fms monoclonalantibodies (Oncogene Sciences, Inc.). The dot blot assay was usedthroughout the purification to identify fractions containing rhsM-CSF.Enriched fractions were pooled, made 0.8 M in ammonium sulfate, adjustedto pH 7.0 and loaded onto a Phenyl TSK-5-PW HPLC column (7.5×75 mm)(BioRad). The column was eluted at 1 ml/min with a decreasing ammoniumsulfate gradient over 45 minutes, peak fractions were pooled andconcentrated 10-fold with a stir cell concentrator using a YM30 membrane(Amicon). The retentate was chromatographed with FG30000XL sizeexclusion column (DU PONT, Wilmington, Del.) using a mobile phase aphosphate-buffered saline (PBS) at 3 ml/min. The purified receptor waspooled, concentrated to 1 mg/ml as above and stored at 4° C. Thisprocess recovered 650 μg of rhsM-CSFR, purified 200-fold. Thepreparation was about 95% homogeneous as assayed by SDS-PAGE stainedwith Coomassie Blue.

EXAMPLE 6 Crystallization of M-CSF/Soluble M-CSF Receptor Complex

To crystallize the M-CSF/rhsM-CSFR complexes, glass microscope slidesare prepared as described in Example 2. The M-CSF composition used isincubated with a purified soluble form of the M-CSF receptor, truncatedat a residue before the transmembrane region, to form an M-CSF/receptorcomplex. In certain cases, the rhsM-CSFR is deglycosylated prior to thesize exclusion step by incubation with N-glycanase (Genzyme, CambridgeMass.) according to the manufacturer's instructions. A small quantity ofM-CSF/receptor solution is mixed in each well of the spot plate with acomparable quantity of a drop solution (such as about 20% (v/v) PEG4000, 0.2 M MgCl₂, 0.1 M Tris-HCl (pH 8.5)). The spot plate is thenplaced in a clear plastic sandwich box containing a small amount ofprecipitant solution (such as about 23% PEG 4000, 0.2 M MgCl₂, 0.1 MTris-HCl (pH 8.5)). The box is immediately sealed and stored at roomtemperature.

After a few days, crystalline M-CSF-receptor complex is isolated andredissolved in a solution containing about 50 mM Tris-HCl and is allowedto stand at room temperature. The purified M-CSF-receptor complexcrystallizes from solution to form crystals for X-ray structuralanalysis. To facilitate solution of the crystal structure of suchcomplexes, truncated, non-glycosylated forms of the rhsM-CSFR (describedabove) which retain M-CSF binding ability may be employed to generateM-CSF-receptor complex crystals.

EXAMPLE 7

The biological activity of the non-glycosylated, truncated sequence usedin Examples 2 and 3 was shown to be equal to that of the mature proteinpurified from human urine (Halenbeck, R., et al., Bio/Technology,7:710-715 [1989]). As noted, the resulting crystals had an orthorhombiccrystal lattice in the P2₁ 2₁ 2₁ space group, with cell dimensionsa=33.54, b=65.26, and c=159.63 Å. Intensity data were collected usingimaging plates mounted on a Weissenberg camera modified formacromolecular crystallography at the Photon Factory in Tsukuba, Japan.Native data to a nominal resolution of 2.0 Å, and mercury and platinumderivative data were collected using 1.0 Å radiation. Two crystalsettings were used to collect native data [Rmerge (I)=7.0%, using allmeasurements with I>0.0].

Heavy atom derivatives of M-CSF crystals were prepared by soakingcrystals in heavy atom compounds dissolved in the reservoir solution.Isomorphous and anomalous difference Patterson maps clearly revealed onesite for the mercury and two sites for the platinum derivative.Anomalous and isomorphous phase information as used in initial phaserefinement with the PROTEIN program package. The final figure of meritwas 0.62 (8.0-3.0 Å, 6960 reflections). After solvent flattening, B. C.Wang, Methods Enzymol, 115:90 (1985), two bundles of four alpha helicesrelated by an approximate two-fold axis could be seen in the electrondensity map. Rotation and translation parameters of thisnon-crystallographic axis were refined by a density correlation method,J. M. Cox, J. Mol. Biol. 28:151 (1967). Phases were then iterativelyrefined by molecular averaging and solvent flattening, G. Bricogne, ActaCryst., 32:832 (1976), using an envelope calculated by putting 5 Åspheres around all the atoms in the four helical bundle. Chain tracingand model building were done in the resulting map, (using the programFRODO), T. A. Jones, Methods Enzymol. 115:157 (1985), keeping theoriginal MIR map as a reference.

The starting partial model for refinement contained only a polyalaninebackbone for eight helices making up the two bundles. Positionalrefinement using the program XPLOR, A. T. Brunger, J. Mol. Biol. 203:803(1985), gave an R-factor of 0.49 to 3.0 Å. Phase combination with therefined MIR phases resulted in a map of sufficient quality to allow thetracing of two long loops traversing the four helical bundle and a shortloop connecting two of the helices. Two strong peaks in the density, oneat the top of the first helix, and the second lying directly on themolecular two fold axis, were assigned as disulfide bonded cysteines.The number of residues between these two peaks uniquely identified theposition in the sequence of these cysteines and consequently thesequence of the intervening residues. This initial registration wasconfirmed by the presence of a number of regions of strong densitycorresponding to aromatic side chains in the sequence. Partial modelphase combination using the added loops and those side chains that werevisible allowed the remaining residues to be registered, thusdetermining the overall topology of the molecule. The presence of sevendisulfide bonds in the dimer served as important "tether points" toconfirm the correctness of the tracing.

As shown in FIG. 2, the overall topology of this form of M-CSF is thatof an antiparallel four α-helical bundle, in which the helices runup-up-down-down, unlike the more commonly observed up-down-up-downconnectivity of most four helical bundles. A long crossover connectionlinks helix A to helix B and a similar connection is found betweenhelices C and D.

A striking difference from other cytokines and other four helix bundlestructures is that the truncated M-CSFα forms a disulfide-linked dimer,in which the bundles are linked end-to-end, forming an extremely flat,elongated structure (approximate dimensions 85×35×25 Å) as shown inFIGS. 3 and 4. There are three intramolecular disulfide bonds in eachmonomer (Cys7-Cys90, Cys48-Cys139, Cys102-Cys146) all of which are atthe distal end of the molecule. One interchain disulfide bond(Cys31--Cys31) is located at the dimer interface with thenoncrystallographic two-fold symmetry axis passing through it as shownin FIGS. 3 and 4A and 4B. Mutation experiments indicate that all of thecysteine residues in this form of M-CSF may be necessary for fullbiological activity. The structure described herein suggests that theirrole is primarily structural rather than being related to receptorrecognition.

Appendix 1 provides the three-dimensional structure of the truncatedrecombinant M-CSFα dimer as identified by the alpha-carbon positions ofthe amino acid residues in the sequence. The five carboxy terminal aminoacids of each polypeptide of the dimer were not included. As will berecognized to those of skill in the art, the information in Appendix 1is provided in the format used by the Brookhaven Protein Data Bank.

As shown, the molecule has an unusual topology which identifiesimportant regions of M-CSF with regard to M-CSF receptor binding.Specific residues in helices A, C, and D appear to be involved in thespecificity of the interaction. Altering solvent accessible residues inthese regions by site directed mutagenesis to increase or decreaseside-chain interactions with the receptor may be useful to generateM-CSF agonists or antagonists. For example, changing one or morehistidines to non-hydrogen-donor amino acids of similar size may createan M-CSF with altered receptor binding ability.

EXAMPLE 8 Preparation of M-CSF Heterodimers

Purification of M-CSF Monomers

E. coli harboring the pL-M-CSF vector for NΔ3CΔ158M-CSFα described inU.S. Pat. No. 4,929,700 or NΔ3CΔ221 M-CSFβ C157S, C159S (Kawasaki, etal., in Colony-Stimulating Factors, Dexter, T., Garland, J., and Testa,N., eds. [1990]) were grown in 1 liter of minimal salts mediumcontaining glucose and the appropriate antibiotic. Expression of M-CSFwas induced by shifting the temperature to 39° C. for 4 hr. followingthe addition of casamino acids to 2%. The cells were harvested bycentrifugation and lysed by sonication in 50 mM Tris (pH 8.5), 10 mMEDTA. The cell debris was recovered by centrifugation, washed with 30%sucrose containing 10 mM EDTA, and a portion of the refractile bodypaste was solubilized in 8 M urea under reducing conditions. Afterincubation at 37° C. for 30 min., the solubilized M-CSF was clarified bycentrifugation and filtration, and then loaded onto a Bio-GelTSK-DEAE-5-PW column (7.5×75 mm) (BioRad Laboratories, Richmond, Calif.)equilibrated in 8 M urea in 10 mM Tris (pH 8.5), 5 mM DTT, 0.5 mM EDTA.The monomeric M-CSF was eluted with a 45-min, 0-0.6 M NaCl gradient. TheM-CSF peak fractions were pooled and concentrated to 10 mg/ml with aCentricon 10 microconcentrator (Amicon).

Formation and Analysis of Active M-CSF Heterodimers

The M-CSF homodimers were refolded by diluting to a proteinconcentration of 0.5 mg/ml in precooled 50 mM Tris (pH 8.5), 5 mM EDTA,2 mM reduced glutathione and 1 mM oxidized glutathione, and thenincubating at 4° C. The heterodimer was refolded by diluting 158 and221F monomer pools to 1 mg/ml in the same buffer. To monitor therefolding, size exclusion high pressure liquid chromatography (SE-HPLC)analysis was performed by immediately injecting reaction samples onto aG3000SW Ultropack (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.)column size-exclusion (7.5×600 mm) equilibrated in PBS (pH 6.8).

Fractionated products were analyzed on reducing SDS-PAGE and stainedwith Coomassie, according to the method of Laemmli, Nature (Canada)227:680-685 (1970). Biological activity was determined using the M-CSFdependent NFS-60 bioassay (See Example 10 below). Antibodyneutralization experiments were carried out by pre-incubatingapproximately 5,000 units of M-CSF dimer with varying dilutions of theneutralizing M-CSF 5H410 Mab (made to refolded E. coli CΔ 150 M-CSFαdimer) prior to bioassay. (Halenbeck et al, Bio/Technology 7:710 (1989)

The heterodimeric M-CSF product was designed to consist of one chain ofshort clone (from amino acid 4 to 158) and one chain of long clone (fromamino acid 4 to 221). The long-clone chain (221F) also containedsubstitutions of serine for the two non-essential cysteines (at 157 and159) to minimize the possibility of higher-order oligomer formation.

Solubilized refractile bodies of M-CSF 158 and 221F were separatelychromatographed by DEAE-HPLC in 8 M urea. Only one major protein peakeluted in each case, and the peak fractions were pooled, based on ananalysis of purity by non-reducing SDS-PAGE and Coomassie staining (datanot shown). The resulting monomer was over 90% pure in each case. Themonomers were separately concentrated to 10 mg/ml, diluted in refoldingbuffer, and refolded at 4° C.

To compare the rates of dimerization of short- and long-clone M-CSF, 20μl of each refolding reaction was injected on a SE-HPLC column at 0, 2,18 and 72 hr. The amount of dimeric M-CSF formed was determined from thepeak area at the molecular weight expected for dimer. In both refoldingreactions the M-CSF was mostly equilibrated to monomer at t=0 and hadbecome about 40% dimeric by 2 hr and nearly 75% dimeric by 18 hr. Thesimilarity of the ratio of dimer to monomer between the refolded 158 and221F strongly suggests that the rate of dimer formation is the same forlong- and short-clone M-CSF. Thus, when equal moles of 158 and 221F arepresent in a refolding reaction, the final relative ratios of 158homodimer to 221F homodimer to 158/221F heterodimer are predicted to be1:1:2 (Similar distributions have been observed in vivo for isozymes oflactate dehydrogenase.)

Biological Activity of Refolded Homodimers and Heterodimers

The biological activity of the refolded homodimers and heterodimersdescribed above, was examined using the in vitro M-CSF-dependent NFS-60bioassay (See Example 10 below). FIG. 5 shows the result of thesestudies. These SE-HPLC and biological activity profiles analyzed after72 hr of refolding, show that the heterodimer displays activity verysimilar to that of the two homodimers. Given that the separation of theheterodimer from the homodimers was nearly complete, it can be concludedthat the heterodimer is fully biologically active in vitro.

To verify that the M-CSF protein eluting from these columns at thepredicted heterodimeric position (between the two homodimers) actuallydid consist of equal moles of short- and long-clone monomers, analysisof a preparative purification of the 158/221F heterodimer was carriedout. Phenyl-HPLC was performed as described above and was shown tocompletely resolve the heterodimer from the 158 and 221F homodimers, asseen in FIG. 6.

Preparative Purification of M-CSF Heterodimers

The refolded M-CSF was adjusted to pH 7.0 with 1 N HCl, and ammoniumsulfate was added to 1.2 M. The protein was loaded onto a Bio-GelTSK-Phenyl-5-PW column (7.5×75 mm) (BioRad, Richmond, Calif.)equilibrated in 1.2 M ammonium sulfate, 100 mM phosphate (pH 7.0). TheM-CSF was eluted with a decreasing gradient of ammonium sulfate from 40%to 80% buffer B (10 mM phosphate, pH 7.0) in 24 min.

Reducing and non-reducing SDS-PAGE showed that internal controls (the158 and 221F dimers) were purified to approximately 95% homogeneity bythis column, and each consisted of the single expected monomeric band.The gel analysis also showed that the heterodimer was purified toapproximately 95% homogeneity and that it consisted of equivalentamounts of 158 and 221F monomers. Recovery of purified 158/221Fheterodimer from refractile body paste to final product, was greaterthan 15%.

The bioactivity of the dimeric M-CSF species was determined and, whencompared to the A₂₈₀ profile in FIG. 6, confirms the finding that theheterodimer is fully active. The specific activity of the 158/221Fheterodimer, calculated using the peak fraction, was 8.0×10⁷ units/mg,compared to 9.0×10⁷ and 6.8×10⁷ units/mg for 158 and 221F homodimers,respectively.

The biological activity of all three dimer species was neutralized tothe same extent in serial dilution neutralization experiments using the5H410 M-CSF Mab in the NFS-60 bioassay. This antibody also neutralizes"naturally refolded" Chinese hamster ovary cell (CHO)-expressed M-CSF ina similar fashion. This result further suggests that the refoldedconformation of the new M-CSF heterodimer is essentially native-like, atleast with regard to the region within the first 150 amino acids that isresponsible for in vitro activity.

EXAMPLE 9 Selection of Amino Acid Substitutions in M-CSF Based onCrystallographic Data

The X-ray crystallographic data described above provided sufficientstructural information regarding M-CSF to be able to identify a limitedsubset of the amino acids in the protein that are likely to be crucialfor M-CSF receptor binding and biological activity and thus whichrepresented likely candidates for mutagenesis with the ultimate goal ofproviding M-CSF muteins having altered biological activity (i.e.,agonists or antagonists). Based on this information, several criteriawere used to generate a list of possible target amino acids forsubstitution.

The first criterion was solvent exposure or solvent accessibility, whichrefers to amino acids residues at the surface of the protein. Residueshaving a solvent accessible surface area of greater than about 0.25 andpreferably greater than about 0.4 are preferred based on normalizationof the surface area of the amino acid accessible when in the trypeptidegly-x-gly (Kabsch, W. et al., Biopolymers 22:2577 (1983)). Residues werechosen which do not interact with other parts of the protein such as thedimer interface in order to maintain the relative orientation ofmonomers and to avoid disturbing the process of protein folding. Stillanother criterion used in certain instances in selecting candidate aminoacid substances is the relationship of the residues to correspondingresidues in mouse M-CSF. Another important selection criterion was thatthe substitutions be non-conservative so as to attempt to disruptpossible hydrogen bonding or hydrophobic interactions with M-CSFreceptor residues.

Table 1 lists exemplary amino acid residues and exemplary substitutions.Using the criteria for selecting candidates for substitutions set forthabove, those of ordinary skill in the art may readily ascertain otherpossible candidates for substitution.

                  TABLE I                                                         ______________________________________                                        Candidate Substitutions                                                            Wild Type Amino Acid and Location                                                                   Substitutions                                      ______________________________________                                        His (H) 15             Ala(A) or Leu(L)                                         Gln (Q) 17 Ala(A) or Glu(E)                                                   Gln (Q) 79 Ala(A) or Asp(D)                                                   Arg (R) 86 Gln(E) or Asp(D)                                                   Glu (E) 115 Ala(A)                                                            Glu (E) 41 Lys(K) or Arg(R)                                                   Lys (K) 93 Ala(A) or Glu(E)                                                   Asp (D) 99 Lys(K) or Arg(R)                                                   Leu (L) 55 Gln(Q) or Asp(N)                                                   Ser (S) 18 Ala(A) or Lys(K)                                                   Gln (Q) 20 Ala(A) or Asp(D)                                                   Arg (R) 21 Ala(A), Glu(E),                                                     or Asp(D)                                                                    Ile (I) 75 Lys(K) or Glu(E)                                                   Val (V) 78 Lys(K) or Arg(R)                                                   Leu (L) 85 Glu(E) or Asn(N)                                                   Asp (D) 69 Lys(K) or Arg(R)                                                   Asn (N) 70 Ala(A) or Glu(E)                                                   His (H) 9 Ala(A) or Asp(D)                                                    Asn (N) 63 Lys(K) or Arg(R)                                                   Thr (T) 34 Gln(Q) or Lys(K)                                                 ______________________________________                                    

It is not expected that every candidate substitution listed will resultin the production of M-CSF agonists or antagonists (see Example 12below). Rather they represent a non-exclusive list of candidates likelyto result in the production of agonists or antagonists based on theselection criteria set forth above. It should also be noted that even ifa variant does not act as an agonist or antagonist when compared withnative M-CSF, the variant is still useful for conventional uses of theligand (if it retains the same activity as the ligand) or as forexample, a diagnostic reagent.

EXAMPLE 10 Preparation of H9A, H15A M-CSF Muteins

A variety of M-CSF muteins with altered solvent-accessible residues fromregions of the M-CSF mature N terminus and helices A, C, and D wereconstructed using techniques known in the art. For example, twohistidines in the N-terminal/A helix region were changed to alaninethrough site-directed mutagenesis of a truncated form of M-CSFα (encodedby pLCSF158A). Involvement of one of three M-CSF histidine residues inM-CSF receptor interaction was implicated by our observation thatdiethylpyrocarbonate (DEPC) modification of histidines in M-CSF at a1:100 DEPC:histidine ratio (as described in Meth. in Enzymol. 47:431(1977)) significantly reduced bioactivity.

Plasmid DNA pLCSF158A was prepared from the E. coli strain HW22 carryingthe plasmid pLCSF158A (U.S. Pat. No. 4,929,700, Example 6, "E. colistrain HW22 transformed with pJN653 containing the asp₅₉ SCSF/NΔ3CΔ158gene"). The strain was grown in 350 ml R2 media (2× Luria Brothcontaining 1% sodium chloride and no glucose, J. Bact., 74:461 (1957))containing 50 micrograms/ml ampicillin at 30° C. with shaking overnight.Plasmid DNA was prepared from the cells using a Qiagen-tip 100 columnaccording to the manufacturer's directions.

Twenty micrograms of pLCSF158A DNA were digested with 66 units ofHindIII and 66 units of StuI at 37° C. for 3 hr. 20 min. in 200microliters 1× New England Biolabs NEBuffer 2 (New England Biolabs,Beverly, Mass.). The DNA was extracted with phenol and chloroform, thenethanol precipitated. The DNA was treated with one unit of CalfIntestinal Alkaline Phosphatase in 100 microliters of 1×Dephosphorylation Buffer, supplied by Boehringer Mannheim (Indianapolis,Ind.), at 37° C. for 30 min. An additional unit of Calf IntestinalAlkaline Phosphatase was added to the reaction and incubation wascontinued at 50° C. for 1 hr.

The resulting DNA was then run on a 1% FMC Bioproducts (Rockland, Me.)Sea KEM® GTG® agarose gel. The 5.7 kb pLCSF158A fragment was cut fromthe gel and purified on Qiagen (Chatsworth, Calif.) Qiaex beadsaccording to the manufacturer's directions.

Polymerase chain reaction (PCR) was then performed and a PCR product wasproduced that contained a mutagenized M-CSF sequence in which histidines9 and 15 (counting from the mature N-terminus) were altered to alanine(generating an H9A, H15A PCR fragment). The 5' portion of the M-CSF genewas amplified from the plasmid pLCSF158A in a PCR reaction using theprimers LF73 and LF74. Details of PCR are provided by Mullis, K. et al.,U.S. Pat. No. 4,683,202; Ehrlich, H., U.S. Pat. No. 4,582,788; Saiki etal., U.S. Pat. No. 4,683,195; Mullis, K. B., Cold Spring Harbor Symp.Quant. Biol. 51:263-273 (1986); Saiki et al., Bio/Technology3:1008-10012 (1985); and Mullis, K. B. et al., Meth. Enzymol 155:335-350(1987), all of which are incorporated herein by reference. The sequencesof these primers are:

    LF73:                                                                           5'-AGGTGTCTCATAGAAAGTTCGGACGCAGGCCTTGTCATGCTCTTCATAATCCTTGG-3' (SEQ ID                                             NO. 1)                                    - LF74:                                                                      5'-CAGGAGAAAGCTTATGTCTGAATATTGTAGCGCCATGATTGGGAGTGGAGCCCTGCAG-3' (SEQ                                              ID NO. 2)                          

The expected PCR product was designed to include 337 bp of pLCSF158Asequence, with the HindIII and StuI sites located at each end of theproduct for cloning, and the histidine codons for His 9 and His 15, CAC,mutated to an alanine codons, GCC.

This product was amplified in four separate PCR reactions eachcontaining 100 ng of pLCSF158A DNA, 50 pmoles LF73, 50 pmoles LF74, 37.5μM dNTPS, 5% glycerol, 1× Perkin Elmer Cetus PCR Buffer, and 2.5 unitsof Perkin Elmer Cetus AmpliTaq® DNA Polymerase in a 100 microlitervolume. The amplification was carried out in a Perkin Elmer Cetus DNAthermocycler. Before adding the AmpliTaq®, the reactions were brought to95° C. The amplification was carried out for 25 cycles ramping to adenaturation temperature of 95° C. in 1 sec., denaturing at 95° C. for 1min.; ramping to an annealing temperature of 68° C. in 1 sec., annealingat 68° C. 1 min.; ramping to an extension temperature of 72° C. in 30sec., extending at 72° C. 1 min., 30 sec. Final extension was carriedout at 72° C. for 10 min.

Five microliters of each reaction were run on a 3% agarose gel (1.5% FMCBioproducts SeaKem® GTG® agarose, 1.5% FMC Bioproducts NuSeive® GTG®agarose in Tris-Borate buffer) (FMC Bioproducts, Rockland, Me.). Gelswere then stained with ethidium bromide. For each reaction, a major bandwas visible at approximately 337 bp.

The four reactions were pooled, extracted with phenol and chloroform,precipitated with ethanol, resuspended and digested with 250 units ofStuI in a final volume of 500 microliters 1× NEBuffer 2 at 37° C. for 2hr., 500 units of HindIII were added to the reaction, the volumeincreased to 1 ml in 1× NEBuffer 2 and digestion was continued at 37° C.for an additional 2.5 hr. The DNA was electrophoresed on a 3% agarosegel. The 300 bp digested product was cut from the gel and purified onQiagen Qiaex beads according to the manufacturer's directions.

Approximately 68 ng of the HindIII/StuI digested PCR product was thenligated to approximately 28 ng of the 5.7 kb HindIII/StuI digestedpLCSF158A vector DNA at an insert-to-vector ratio of approximately 5:1.Ligation was carried out with 1 unit of Boehringer Mannheim T4 DNAligase in 1× ligation buffer, supplied by the manufacturer, in a20-microliter volume at 16° C. overnight. As a control 28 ng of the 5.7kb HindIII/StuI digested pLCSF158A vector DNA was ligated to itselfunder the same conditions with no insert present.

Half of each ligation mixture was used to transform competent E. coliDG116 (ATCC#53606) cells using a protocol similar to the calciumchloride procedure described in Molecular Cloning a Laboratory ManualManiatis et al., Cold Spring Harbor Laboratory (1982). Transformed cellswere allowed to express at 30° C. with no selection for 90 min., platedon R2-4 (10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 drops antifoam A,4 ml 50% glucose and 15 g agar in 1 liter) plates containing 50micrograms/ml ampicillin. The plates were incubated at room temperature72 hr. One fourth of each transformation was plated. For the ligationcontaining the insert, 66 ampicillin resistant colonies appeared on theplates. For the ligation with no insert, no colonies appeared.

One of these colonies, designated strain TAF172-2, was picked andcultured in 350 ml R2 broth with 50 micrograms/ml ampicillin at 30° C.with shaking overnight. A frozen stock in 40% glycerol was made fromthis culture and stored at -70° C. DNA was isolated from the cultureusing Qiagen-tip 100 columns as described above.

The purified DNA, pTAF172-2, was sequenced using the di-deoxy method andshown to contain the sequence of pLCSF158A coding for M-CSF NΔ3CΔ158with His 9 and His 15 mutated to encode Ala.

The M-CSF mutein NΔ3CΔ158 H9A, H15A encoded by pTAF172-2 was expressed,purified, refolded to form dimeric protein and assayed essentially asdescribed in U.S. Pat. No. 4,929,700 Example 5, using 8M urea as adenaturant and in the DEAE purification step.

The N-terminal sequence of the purified mutein was determined through 20cycles, using a standard automated Edman degradation method, and shownto be identical to that of the parental NΔ3CΔ158 M-CSFα referenceprotein except that His 9 and His 15 had been altered to Ala. Proteinconcentration was determined using A280 and an extinction co-efficientof 0.68.

The purified mutein dimers were subjected to bioassay using NFS-60 cellswhich is an M-CSF dependent cell line which forms colonies in thepresence of active M-CSF. Standards and purified mutein samples wereserially diluted 1:2 in RPMI media (10% fetal bovine serum) in a 50microliter volume in 96-well microtiter plates. 50 microliters NFS-60cells (ATCC NO. CRL 1838), maintained in 4000 U/ml M-CSF, washed 2×, anddiluted to a concentration of 1×10⁵ cell/ml, were added to each samplewell. Plates were incubated at 37° C., 5% CO₂ for 44 h. 5 mg/ml MTT dyewas added to each sample and incubation continued for 4 h. 20% SDS wasadded to each sample. The plates were wrapped in foil and leftovernight. The absorbance of each sample was read at 570 nm and comparedto a standard of known M-CSF concentration. The H9A, H15A mutein showeda specific activity of 7.6×10³ U/mg compared to 6.9×10⁷ U/mg for theparental M-CSF NΔ3CΔ158 reference in the same assay. This represents anearly 10,000 fold reduction in biological activity for the mutein. Thesame M-CSF mutein preparation was shown to have greatly decreased M-CSFreceptor-binding ability using the NFS-60 receptor competition assaydescribed in this example. Because the H9A, H15A M-CSF mutein wasotherwise not significantly different from the parental M-CSFα,including crystallizability and space group (see Example 12) we believethe decrease in biological activity is not due to gross deformation ofstructure but reflects an alteration in important M-CSF receptorcontacts.

Using essentially the same methodology, two M-CSF muteins contactingsingly substituted histidines at residues 9 and 15 were generated (e.g.H9A and H15A). The H9A construct utilized LF80:

5'-CAGGAGAAAGCTTATGTCTGAATATTGTAGCGCCATGATTGGGAGTGGACACCTGCAG-3' (SEQ IDNO 3); and LF73 (described in this example) as the PCR primers. The H15Aconstruct utilized LF81:

5'-CAGGAGAAAGCTTATGTCTGAATATTGTAGCCACATGATTGGGAGTGGAGCCCTGCAG-3' (SEQ IDNO.4); and LF 73 (described in this example) as PCR primers. Biologicalassay of the purified muteins immediately following the refolding stepdescribed above showed approximate biological specific activities asfollows: 4×10⁶ U/mg for H9A and less than 3×10³ U/mg for H15A, in anassay which the parental M-CSF construct displayed 8×10⁷ U/mg. Thisinformation, combined with that described above, suggests that H15A aswell as possible H9A represents contacts that are important for M-CSFreceptor binding. Nearby solvent accessible residues such as Y6 and S13(see also Table 1) may also represent M-CSF receptor contact residues.Non-proteinaceous mimics of the side chains of H9, H15, and nearbysolvent accessible side chains may represent M-CSF agonists orantagonists. Such residues should be left unchanged in M-CSF muteinconstructs designed to retain full M-CSF receptor binding but to haveM-CSF antagonist properties because they lack significant M-CSFbioactivity. Homodimers of muteins that retain full receptor-bindingability and display significantly reduced bioactivity should representM-CSF antagonists. M-CSF muteins that are greatly decreased in bothM-CSF bioactivity and receptor binding ability (such as H15A) maygenerally be useful in M-CSF immunoassay applications and mightrepresent useful therapeutic agents for patients having auto-antibodiesto M-CSF.

EXAMPLE 11 Preparation of Q20A, V78K M-CSF Muteins

Using essentially the same methodology described in Example 10, a doublemutant of M-CSF (Q20A, V78K) was constructed to test the importance ofsolvent accessible residues in the central portion of helices A and C.The following PCR primers were used.

    LF63:                                                                           5'-AGGAGAAAGCTTATGTCTGAATATTGTAGCCACATGATTGGGAGTGGACACCTGCAGT(SEQ ID                                              NO. 5)                                    CTCTGGCTCGGCTG-3'                                                              - LF64:                                                                      5'-GGACGCAGGCCTTGTCATGCTCTTCATAATCCTTGGTGAAGCAGCTCTTCAGCCTCAA(SEQ ID                                              NO. 6)                                    AGAGAGTTCCTGCAGCTGTTTAATGGC-3'                                          

The resulting mutein was expressed, refolded, purified, and assayed asdescribed in Example 8. The specific biological activity was 1.4×10⁷U/mg, approximately 8-10 fold lower than that of the parental M-CSFαreference standard. The receptor binding activity of this mutein wasalso decreased.

This result again supports the prediction of over crystallographic studyof truncated M-CSFα which concluded that important M-CSF receptorcontact residues exist among the solvent accessible residues in helicesA and/or C and/or D. Certain of these mutations will, as we have shown,have lower biological activity and lower M-CSF receptor-binding ability.Some may have lower biological activity without a decrease inreceptor-binding ability. Some may have increased biological activityand receptor binding ability, and some may have no affect on either.

Two examples of the latter are Q17A, R21A (produced using PCR primersLF72:

5'-TTGTAGCCACATGATTGGGAGTGGACACCTGGCGTCTCTGCAGGCGCTGATTGAC-3 and

LF73 (described in Example 9) and E115A, N119A (produced using LF75:

5'-CATGACAAGGCCTGCGTCCGAACTTTCTATGAGACACCTCTCCAGTTGCTGGCGAAGGTCAAGGCTGTCTTTAATG-3' (SEQ ID NO. 7); and

LF79: 5'-GGATCAGGATCCCTCGGACTGCCTCTC-3' (SEQ ID NO. 8)).

Both of these constructs changed side chain properties ofsolvent-accessible amino acids in the areas of interest but did notaffect biological specific activity, compared to the parental referencemolecule. These results indicate that residues Q17, R21, E115, and N119do not need to be altered in muteins designed to have M-CSF agonist orantagonist activity. In fact, to minimize the likelihood of antibodyformation to potentially administered M-CSF-based proteinaceous drugs,it is desirable to retain the solvent-accessible parental M-CSF residues(to resemble the native molecule) whenever possible.

The retained activity of the muteins including changes at Q17, R21,E115, and N119 does not rule out large affects on activity contributedby nearby residues (such as H15). In fact, the regions we have alteredare predicted by the crystal structure to be important for receptorbinding and/or signaling. Antagonistic M-CSF muteins may require use ofmultiple residue changes per mutein or use of heterodimeric moleculescontaining one or more mutations in each polypeptide chain, since M-CSFresidues important in receptor signaling are believed to be composed ofdiscontinuous regions of M-CSF.

EXAMPLE 12 Formation of M-CSF Heterodimers Having DecreasedReceptor-Binding Ability and/or Decreased Biological Specific Activity

M-CSF can be folded in vitro to generate fully active heterodimers, asshown in Example 8. By making heterodimers of M-CSF which incorporateM-CSF muteins with altered M-CSF signaling ability, it should bepossible to generate antagonists of M-CSF useful for treatment ofpatients with M-CSF mediated diseases. To generate a heterodimercontaining one subunit of M-CSFα NΔ3CΔ158 H9A, H15A and one subunit ofM-CSFβ NΔ3CΔ221 C157S, C159S, each mutein was expressed in E. coli andpurified separately by DEAE Sepharose under denaturing and reducingconditions as described in Example 8. The two muteins subunits weremixed together prior to refolding to generate a solution containing afinal mutein molar ratio of 1:1, then this solution was diluted to 0.2mg/ml with refolding buffer as described in Example 8. Followingrefolding, the heterodimeric molecule was separated from the homodimersby two consecutive passes over a Phenyl TSK-5-PW HPLC column asdescribed in Example 8. No contaminants were detected when the purifiedheterodimer preparation was examined by non-reduced SDS-PAGE or sizeexclusion HPLC using a BioSil SEC250 column (BioRad).

The purified heterodimer was submitted to the NFS60 cell based bioassaydescribed in Example 8. The calculated specific activity was 2.9×10⁶U/mg which correlated to a 35-fold reduction as compared to the activityof the parental M-CSF heterodimer described in Example 10. The relativebinding affinity to cell surface M-CSF receptor was measured byradioligand displacement in which the displacement of ¹²⁵ I-M-CSF froman M-CSF receptor by an M-CSF mutein was measured using methods wellknown in the art. In brief, the following were added in a final volumeof 100 μl in each well of a 96-well cell culture plate: approximately80,000 cpm of purified recombinant human M-CSF labeled with ¹²⁵ I(usingIodobeads as described by the manufacturer, Pierce, Rockford, Ill.),300,000 NFS-60 cells that had been washed and then grown for 18 hours ingrowth medium minus the normal maintenance level of M-CSF, plusunlabeled M-CSF that had been serially diluted in the same medium. Theplates were incubated at 4° C. for 20 hours and the cells were collectedon glass-fiber filters. Maximum binding was measured in the absence ofunlabeled M-CSF and non-specific binding was measured in the presence of1000-fold greater concentration of unlabeled M-CSF (compared to labeledM-CSF). The concentration of M-CSF required to inhibit 50% of thelabeled M-CSF binding to the cells (IC₅₀) was used to determinedifferences in affinity. Results are expressed as percent displacementof radioactive M-CSF versus mutein concentration (FIG. 7). The IC₅₀ ofthe heterodimer (FIG. 7 closed squares) was increased 30-fold to about500 pM as compared to an IC₅₀ of about 17 pM for M-CSFα NΔ3CΔ158 (158)(FIG. 7 closed circles). The similarity between the reduction inspecific activity and receptor affinity of the heterodimer indicatesthat the reduction in bioactivity was due to decreased receptor-bindingability. Similarly, the binding affinities of the Q20A, V78KF (FIG. 7open circles) and H9A, H15A (open squares) muteins were also measured inthis radioligand displacement assay (FIG. 7). The Q20A, V78K mutein hadan IC₅₀ of about 100 pM and the H9A/H15A mutein has an IC₅₀ of about 1μM, correlating to decreased binding affinities of 5-fold and50,000-fold, respectively. For each mutein, the reduction in receptoraffinity was similar to the reduction in specific activity, againindicating that the reduction in bioactivity was due to reducedreceptor-binding ability.

EXAMPLE 13 Crystallization and Characterization of M-CSF H9A, H15AMuteins

The H98, H15A mutein described in Example 10 was crystallized using thehanging drop method described in Examples 1 and 2 using the followingbuffer conditions: 30% polyethylene glycol 4000; 100 mM Li₂ SO₄ ; and100 mM Tris pH 8.5. The crystals produced under these conditions wererhombohedral prisms having dimensions of 0.7 mm×0.2 mm×0.2 mm. X-raycrystallographic analysis using precession photographs showed crystalsin the P2₁ 2₁ 2₁ space group with cell dimensions of a=33.99, b=65.37,c=159.90, d=90, e=90, and f=90 angstroms and diffract to a nominalresolution of 3 angstroms. These physical properties are essentially thesame as those observed for the parental NΔ3CΔ158 M-CSFα molecule andsuggests that the biological effects of the H9A, H15A alterations arenot the consequence of gross global alterations in M-CSF structure, butrather are the result of altered side chains that are important ininteracting with the M-CSF receptor. Alteration of those histidine sidechains may have affected receptor binding by changing atoms thatinteract with, stabilize or facilitate receptor binding or changes inreceptor conformation. Changes such as H15A may also have affected thesefunctions by altering the position of the nearby side chain in M-CSF,most likely in the A and/or C helix regions.

The foregoing examples are presented by way of example and are notintended to limit the scope of the invention as set forth in theappended claims.

    __________________________________________________________________________    ATOM                                                                               10 CA SER  4  63.753                                                                             80.590                                                                           222.385                                                                           1.00                                                                              58.89                                        ATOM  13 CA GLU  5 64.883  77.664 219.972 1.00 59.56                          ATOM  23 CA TYR  6 62.285  76.840 217.324 1.00 54.54                          ATOM  37 CA CYS  7 63.509  80.109 215.834 1.00 5A.96                          ATOM  44 CA SER  8 66.853  79.529 214.160 1.00 54.41                          ATOM  52 CA HIS  9 65.466  77.798 211.053 1.00 55.41                          ATOM  65 CA MET  10 61.857  78.767 211.073 1.00 50.66                         ATOM  74 CA ILE  11 62.173  80.905 207.970 1.00 48.31                         ATOM  83 CA GLY  12 63.487  78.618 205.354 1.00 52.83                         ATOM  88 CA SER  13 64.609  79.967 201.952 1.00 53.13                         ATOM  96 CA GLY  14 61.665  78.009 200.514 1.00 46.05                         ATOM  101 CA HIS  15 59.455  80.924 201.933 1.00 40.25                        ATOM  114 CA LEU  16 62.210  83.200 200.992 1.00 41.58                        ATOM  123 CA GLN  17 62.153  82.266 197.328 1.00 46.17                        ATOM  135 CA SER  18 58.378  81.868 197.227 1.00 46.55                        ATOM  143 CA LEU  19 58.280  85.544 198.199 1.00 44.75                        ATOM  152 CA GLN  20 60.814  86.044 195.458 1.00 39.32                        ATOM  164 CA ARG  21 58.457  84.718 192.736 1.00 36.91                        ATOM  181 CA LEU  22 55.610  86.694 194.252 1.00 38.27                        ATOM  190 CA ILE  23 57.676  89.822 193.465 1.00 34.84                        ATOM  199 CA ASP  24 58.677  88.274 190.086 1.00 31.50                        ATOM  208 CA SER  25 55.086  87.802 188.978 1.00 31.73                        ATOM  216 CA GLN  26 54.284  91.540 189.154 1.00 33.24                        ATOM  228 CA MET  27 53.961  93.634 186.082 1.00 34.82                        ATOM  237 CA GLU  28 56.227  96.566 186.089 1.00 36.54                        ATOM  247 CA THR  29 53.758  99.303 186.920 1.00 44.80                        ATOM  256 CA SER  30 54.590 102.747 188.323 1.00 49.40                        ATOM  264 CA CYS  31 51.427 103.423 190.046 1.00 43.03                        ATOM  271 CA GLN  32 52.166 104.336 193.613 1.00 33.74                        ATOM  283 CA ILE  33 50.430 102.961 196.634 1.00 32.48                        ATOM  292 CA THR  34 50.240 104.213 200.133 1.00 38.94                        ATOM  301 CA PHE  35 51.291 102.173 203.199 1.00 39.16                        ATOM  313 CA GLU  36 52.707 102.849 206.761 1.00 32.09                        ATOM  323 CA PHE  37 56.073 101.358 207.299 1.00 25.78                        ATOM  335 CA VAL  38 59.082 101.751 209.490 1.00 34.46                        ATOM  343 CA ASP  39 61.044 104.798 208.714 1.00 44.03                        ATOM  352 CA GLN  40 64.648 103.690 208.314 1.00 54.20                        ATOM  364 CA GLU  41 65.924 107.142 209.332 1.00 52.01                        ATOM  374 CA GLN  42 63.934 107.629 212.631 1.00 44.26                        ATOM  386 CA LEU  43 64.770 104.161 213.955 1.00 45.54                        ATOM  395 CA ALA  44 68.126 102.952 212.789 1.00 51.53                        ATOM  401 CA ASP  45 69.175 100.197 215.232 1.00 47.                          ATOM  410 CA PRO  46 68.861  97.054 213.098 1.00 45.27                        ATOM  417 CA VAL  47 67.352  94.587 215.613 1.00 39.92                        ATOM  425 CA CYS  48 64.862  97.220 216.692 1.00 36.44                        ATOM  432 CA TYR  49 64.089  98.158 213.105 1.00 37.39                        ATOM  446 CA LEU  50 63.436  94.474 212.189 1.00 36.15                        ATOM  455 CA LYS  51 61.603  94.029 215.472 1.00 38.66                        ATOM  468 CA LYS  52 59.322  96.862 214.367 1.00 41.72                        ATOM  481 CA ALA  53 59.229  96.177 210.534 1.00 35.81                        ATOM  487 CA PHE  54 58.096  92.701 211.432 1.00 40.82                        ATOM  499 CA LEU  55 55.236  93.733 213.614 1.00 42.59                        ATOM  508 CA LEU  56 54.205  95.869 210.697 1.00 44.24                        ATOM  517 CA VAL  57 54.596  93.257 207.992 1.00 35.10                        ATOM  525 CA GLN  58 51.646  91.431 209.628 1.00 39.91                        ATOM  537 CA ASP  59 49.277  94.254 208.663 1.00 39.60                        ATOM  546 CA ILE  60 50.755  94.729 205.185 1.00 37.05                        ATOM  555 CA MET  61 50.303  90.932 204.676 1.00 33.86                        ATOM  564 CA GLU  62 46.766  90.931 206.028 1.00 48.10                        ATOM  574 CA ASP  63 45.602  94.198 204.286 1.00 49.51                        ATOM  583 CA THR  64 47.659  95.244 201.187 1.00 42.65                        ATOM  592 CA MET  65 49.106  92.155 199.603 1.00 37.90                        ATOM  601 CA ARG  66 45.646  91.249 198.376 1.00 39.50                        ATOM  618 CA PHE  67 45.516  88.245 195.824 1.00 35.13                        ATOM  630 CA ARG  68 42.655  86.013 194.326 1.00 44.68                        ATOM  647 CA ASP  69 41.727  83.454 196.865 1.00 47.42                        ATOM  656 CA ASN  70 43.117  80.057 195.984 1.00 44.47                        ATOM  667 CA THR  71 45.873  81.088 193.518 1.00 34.80                        ATOM  676 CA PRO  72 49.607  80.251 194.114 1.00 29.78                        ATOM  683 CA ASN  73 50.307  83.745 195.418 1.00 28.47                        ATOM  694 CA ALA  74 47.469  83.922 197.953 1.00 23.24                        ATOM  700 CA ILE  75 48.690  80.431 199.144 1.00 22.87                        ATOM  709 CA ALA  76 52.212  81.892 199.669 1.00 29.47                        ATOM  715 CA ILE  77 50.816  84.796 201.668 1.00 37.21                        ATOM  724 CA VAL  78 48.902  82.158 203.857 1.00 33.69                        ATOM  732 CA GLN  79 52.207  80.382 204.466 1.00 33.36                        ATOM  744 CA LEU  80 54.040  83.518 205.449 1.00 32.55                        ATOM  753 CA GLN  81 51.152  84.517 207.743 1.00 35.07                        ATOM  765 CA GLU  82 51.541  81.115 209.443 1.00 37.26                        ATOM  775 CA LEU  83 55.367  81.348 209.619 1.00 35.10                        ATOM  784 CA SER  84 54.960  84.840 211.066 1.00 39.64                        ATOM  792 CA LEU  85 52.608  83.541 213.704 1.00 42.55                        ATOM  801 CA ARG  86 54.984  80.903 215.121 1.00 42.96                        ATOM  818 CA LEU  87 57.916  83.317 214.670 1.00 45.11                        ATOM  827 CA LYS  88 56.504  85.876 217.138 1.00 50.71                        ATOM  840 CA SER  89 57.621  83.610 219.825 1.00 54.58                        ATOM  848 CA CYS  90 61.211  84.524 219.014 1.00 46.79                        ATOM  855 CA PHE  91 60.235  88.070 219.743 1.00 46.37                        ATOM  867 CA THR  92 59.619  89.812 223.026 1.00 52.01                        ATOM  876 CA ALA  93 56.916  92.445 223.523 1.00 56.09                        ATOM  882 CA ASP  94 57.914  96.123 223.998 1.00 59.35                        ATOM  891 CA TYR  95 55.685  99.100 225.194 1.00 66.22                        ATOM  905 CA GLU  96 52.401 100.066 223.612 1.00 65.51                        ATOM  915 CA GLU  97 53.343 103.890 223.182 1.00 62.60                        ATOM  925 CA HIS  98 56.046 102.712 220.836 1.00 58.47                        ATOM  938 CA ASP  99 53.422 100.733 218.887 1.00 59.97                        ATOM  947 CA LYS 100 52.950 103.470 216.162 1.00 57.00                        ATOM  960 CA ALA 101 56.259 105.270 216.975 1.00 48.82                        ATOM  966 CA CYS 102 58.861 105.450 214.154 1.00 42.41                        ATOM  973 CA VAL 103 56.175 104.773 211.621 1.00 33.87                        ATOM  981 CA ARG 104 55.993 106.851 208.456 1.00 42.76                        ATOM  998 CA THR 105 53.577 106.853 205.501 1.00 38.00                        ATOM 1007 CA PHE 106 54.931 105.944 202.136 1.00 33.48                        ATOM 1019 CA TYR 107 53.853 106.546 198.455 1.00 34.28                        ATOM 1033 CA GLU 108 55.765 103.906 196.543 1.00 33.88                        ATOM 1043 CA THR 109 55.266 101.309 193.854 1.00 36.94                        ATOM 1052 CA PRO 110 53.745  97.857 194.657 1.00 29.84                        ATOM 1059 CA LEU 111 57.132  96.374 193.161 1.00 35.66                        ATOM 1068 CA GLN 112 58.991  98.756 196.160 1.00 33.48                        ATOM 1080 CA LEU 113 56.612  97.691 198.912 1.00 24.26                        ATOM 1089 CA LEU 114 57.253  93.932 198.091 1.00 30.                          ATOM 1098 CA GLU 115 61.063  94.385 197.984 1.00 39.68                        ATOM 1108 CA LYS 116 60.805  95.824 201.526 1.00 39.24                        ATOM 1121 CA VAL 117 58.669  92.840 202.743 1.00 32.46                        ATOM 1129 CA LYS 118 61.312  90.620 201.111 1.00 37.34                        ATOM 1142 CA ASN 119 64.231  92.316 202.959 1.00 39.46                        ATOM 1153 CA VAL 120 62.470  92.033 206.370 1.00 32.07                        ATOM 1161 CA PHE 121 61.935  88.268 206.034 1.00 26.35                        ATOM 1173 CA ASN 122 65.404  88.049 204.416 1.00 36.04                        ATOM 1184 CA GLU 123 67.372  89.816 207.138 1.00 40.82                        ATOM 1194 CA THR 124 65.331  88.435 210.122 1.00 41.72                        ATOM 1203 CA LYS 125 66.456  85.000 208.648 1.00 43.07                        ATOM 1216 CA ASN 126 70.010  86.339 208.179 1.00 48.89                        ATOM 1227 CA LEU 127 70.000  87.300 211.903 1.00 47.97                        ATOM 1236 CA LEU 128 68.315  84.194 213.435 1.00 47.21                        ATOM 1245 CA ASP 129 71.086  82.134 211.864 1.00 50.11                        ATOM 1254 CA LYS 130 73.448  84.146 214.117 1.00 52.04                        ATOM 1267 CA ASP 131 71.442  84.303 217.451 1.00 49.58                        ATOM 1276 CA TRP 132 67.946  82.685 218.322 1.00 53.33                        ATOM 1292 CA ASN 133 67.568  85.178 221.086 1.00 59.61                        ATOM 1303 CA ILE 134 68.572  88.447 219.305 1.00 49.82                        ATOM 1312 CA PHE 135 64.945  89.516 219.045 1.00 54.55                        ATOM 1324 CA SER 136 64.745  89.689 222.796 1.00 62.                          ATOM 1332 CA LYS 137 66.563  93.119 222.569 1.00 54.38                        ATOM 1345 CA ASN 138 64.785  95.734 224.504 1.00 58.71                        ATOM 1356 CA CYS 139 63.902  98.337 222.050 1.00 51.21                        ATOM 1363 CA ASN 140 61.704 100.5  224.157 1.00 50.25                         ATOM 1374 CA ASN 141 64.219 103.338 224.061 1.00 58.30                        ATOM 1385 CA SER 142 65.158 102.992 220.352 1.00 52.18                        ATOM 1393 CA PHE 143 61.498 103.434 219.645 1.00 45.87                        ATOM 1405 CA ALA 144 61.829 106.488 221.831 1.00 47.49                        ATOM 1411 CA GLU 145 64.873 108.022 220.050 1.00 49.94                        ATOM 1421 CA CYS 146 62.489 108.299 217.017 1.00 53.65                        ATOM 1428 CA SER 147 60.005 111.197 217.009 1.00 57.45                        ATOM 1436 CA SER 148 57.163 110.805 214.395 1.00 59.53                        ATOM 1444 CA ALA 149 54.284 108.658 214.945 1.00 59.20                        ATOM 1450 CA GLY 150 51.655 108.783 212.248 1.00 61.95                        ATOM 1455 CA HIS 151 48.764 106.945 210.680 1.00 69.20                        ATOM 1468 CA GLU 152 46.475 107.377 207.803 1.00 76.61                        ATOM 1478 CA ALA 153 45.813 106.188 204.264 1.00 78.85                        ATOM 1492 CA SER 404 43.875  81.916 155.536 1.00 38.68                        ATOM 1495 CA GLU 405 41.939  78.947 156.798 1.00 43.57                        ATOM 1505 CA TYR 406 44.549  78.204 159.422 1.00 41.97                        ATOM 1519 CA CYS 407 43.251  81.196 161.434 1.00 36.99                        ATOM 1526 CA SER 408 41.011  78.917 163.442 1.00 34.02                        ATOM 1534 CA HIS 409 43.988  77.122 165.021 1.00 40.02                        ATOM 1547 CA MET 410 46.155  79.994 165.895 1.00 36.48                        ATOM 1556 CA ILE 411 44.849  80.843 169.353 1.00 37.88                        ATOM 1565 CA GLY 412 44.508  77.692 171.141 1.00 39.09                        ATOM 1570 CA SER 413 43.275  77.480 174.756 1.00 45.21                        ATOM 1578 CA GLY 414 46.813  77.142 176.120 1.00 41.00                        ATOM 1583 CA HIS 415 47.238  80.801 175.208 1.00 38.01                        ATOM 1596 CA LEU 416 44.181  81.554 177.458 1.00 39.12                        ATOM 1605 CA GLN 417 45.501  79.575 180.501 1.00 39.79                        ATOM 1617 CA SER 418 48.547  81.846 180.317 1.00 36.95                        ATOM 1625 CA LEU 419 46.482  85.106 180.212 1.00 33.42                        ATOM 1634 CA GLN 420 44.564  83.708 183.162 1.00 38.37                        ATOM 1646 CA ARG 421 47.839  82.875 185.041 1.00 41.95                        ATOM 1663 CA LEU 422 49.151  86.514 184.405 1.00 33.04                        ATOM 1672 CA ILE 423 45.354  87.864 185.738 1.00 25.14                        ATOM 1681 CA ASP 424 45.824  85.549 188.861 1.00 32.93                        ATOM 1690 CA SER 425 49.394  86.494 189.904 1.00 30.92                        ATOM 1698 CA GLN 426 48.776  90.276 189.999 1.00 29.47                        ATOM 1710 CA MET 427 48.379  91.899 193.372 1.00 3.1.04                       ATOM 1719 CA GLU 428 44.978  93.418 193.655 1.00 42.94                        ATOM 1729 CA THR 429 45.965  96.999 194.423 1.00 48.52                        ATOM 1738 CA SER 430 43.656  99.720 193.224 1.00 48.67                        ATOM 1746 CA CYS 431 46.604 101.354 191.803 1.00 52.3                         ATOM 1753 CA GLN 432 45.813 102.789 188.396 1.00 50.77                        ATOM 1765 CA ILE 433 47.826 103.166 185.221 1.00 51.17                        ATOM 1774 CA THR 434 46.923 105.057 182.065 1.00 52.62                        ATOM 1783 CA PHE 435 46.641 103.293 178.672 1.00 50.53                        ATOM 1795 CA GLU 436 45.167 103.968 175.164 1.00 50.56                        ATOM 1805 CA PHE 437 42.643 101.271 174.193 1.00 46.56                        ATOM 1817 CA VAL 438 39.404 101.233 172.116 1.00 52.14                        ATOM 1825 CA ASP 439 36.342 103.107 173.476 1.00 59.69                        ATOM 1834 CA GLN 440 33.750 100.386 173.243 1.00 63.82                        ATOM 1846 CA GLU 441 30.764 102.682 173.229 1.00 64.79                        ATOM 1856 CA GLN 442 31.810 104.569 170.091 1.00 59.01                        ATOM 1868 CA LEU 443 32.994 101.521 168.139 1.00 55.78                        ATOM 1877 CA LYS 444 30.279  99.137 169.086 1.00 54.82                        ATOM 1890 CA ASP 445 30.862  96.668 166.093 1.00 51.31                        ATOM 1899 CA PRO 446 32.144  93.267 167.135 1.00 47.34                        ATOM 1906 CA VAL 447 34.363  92.592 164.111 1.00 41.42                        ATOM 1914 CA CYS 448 35.680  96.184 163.845 1.00 39.44                        ATOM 1921 CA TYR 449 36.273  96.949 167.607 1.00 40.33                        ATOM 1935 CA LEU 450 38.224  93.580 167.624 1.00 30.87                        ATOM 1944 CA LYS 451 39.989  94.664 164.462 1.00 34.49                        ATOM 1957 CA LYS 452 40.962  97.918 166.163 1.00 35.38                        ATOM 1970 CA ALA 453 41.503  96.317 169.572 1.00 28.                          ATOM 1976 CA PHE 454 44.012  93.845 168.164 1.00 32.56                        ATOM 1988 CA LEU 455 46.236  96.653 166.861 1.00 40.19                        ATOM 1997 CA LEU 456 46.402  98.605 170.114 1.00 42.45                        ATOM 2006 CA VAL 457 46.966  95.358 172.060 1.00 38.09                        ATOM 2014 CA GLN 458 50.298  95.232 170.161 1.00 48.02                        ATOM 2026 CA ASP 459 51.630  98.529 171.587 1.00 41.96                        ATOM 2035 CA ILE 460 50.230  97.686 175.001 1.00 25.82                        ATOM 2044 CA MET 461 52.409  94.542 175.085 1.00 33.69                        ATOM 2053 CA GLU 462 55.702  96.253 173.969 1.00 45.36                        ATOM 2063 CA ASP 463 55.069  99.093 176.249 1.00 48.41                        ATOM 2072 CA THR 464 52.771  98.555 179.186 1.00 41.63                        ATOM 2081 CA MET 465 52.743  94.854 180.171 1.00 42.05                        ATOM 2090 CA ARG 466 56.333  94.928 181.338 1.00 44.99                        ATOM 2107 CA PHE 467 57.618  91.923 183.501 1.00 38.72                        ATOM 2119 CA ARG 468 61.258  90.894 184.513 1.00 50.64                        ATOM 2136 CA ASP 469 63.046  88.868 181.895 1.00 53.46                        ATOM 2145 CA ASN 470 62.896  85.096 181.832 1.00 52.84                        ATOM 2156 CA THR 471 60.211  84.798 184.479 1.00 47.58                        ATOM 2165 CA PRO 472 57.055  82.652 183.744 1.00 46.82                        ATOM 2172 CA ASN 473 54.971  85.777 183.172 1.00 38.75                        ATOM 2183 CA ALA 474 57.431  87.402 180.828 1.00 37.27                        ATOM 2189 CA ILE 475 57.795  84.219 178.807 1.00 32.85                        ATOM 2198 CA ALA 476 54.021  84.034 178.455 1.00 27.98                        ATOM 2204 CA ILE 477 53.844  87.522 176.756 1.00 30.47                        ATOM 2213 CA VAL 478 56.690  86.495 174.201 1.00 30.40                        ATOM 2221 CA GLN 479 54.381  83.711 173.325 1.00 31.18                        ATOM 2233 CA LEU 480 51.419  86.038 172.893 1.00 27.44                        ATOM 2242 CA GLN 481 53.392  88.551 170.821 1.00 31.59                        ATOM 2254 CA GLU 482 54.572  85.683 168.506 1.00 29.90                        ATOM 2264 CA LEU 483 50.900  84.532 168.340 1.00 22.16                        ATOM 2273 CA SER 484 50.077  88.156 167.451 1.00 29.37                        ATOM 2281 CA LEU 485 52.410  87.880 164.490 1.00 32.70                        ATOM 2290 CA ARG 486 50.865  84.572 163.229 1.00 30.59                        ATOM 2307 CA LEU 487 47.396  86.383 163.343 1.00 31.02                        ATOM 2316 CA LYS 488 48.292  89.399 161.352 1.00 34.90                        ATOM 2329 CA SER 489 47.728  87.397 158.200 1.00 35.68                        ATOM 2337 CA CYS 490 44.112  87.134 159.417 1.00 35.03                        ATOM 2344 CA PHE 491 43.520  90.786 159.602 1.00 42.73                        ATOM 2356 CA THR 492 43.145  93.174 156.663 1.00 56.24                        ATOM 2365 CA ALA 493 44.877  96.467 156.617 1.00 57.56                        ATOM 2371 CA ASP 494 42.318  99.142 157.302 1.00 57.16                        ATOM 2380 CA ALA 495 42.063 102.909 156.516 1.00 62.14                        ATOM 2386 CA GLU 496 45.542 104.198 157.805 1.00 64.30                        ATOM 2396 CA GLU 497 44.589 107.547 159.382 1.00 70.67                        ATOM 2406 CA HIS 498 41.577 105.660 160.752 1.00 75.16                        ATOM 2419 CA ASP 499 44.400 103.329 161.968 1.00 72.11                        ATOM 2428 CA LYS 500 44.773 105.712 164.874 1.00 65.                          ATOM 2441 CA ALA 501 41.040 106.628 165.617 1.00 55.63                        ATOM 2447 CA CYS 502 38.563 105.427 168.336 1.00 53.33                        ATOM 2454 CA VAL 503 41.155 105.639 171.100 1.00 52.46                        ATOM 2462 CA ARG 504 39.866 106.944 174.425 1.00 51.00                        ATOM 2479 CA THR 505 42.588 106.921 177.117 1.00 50.42                        ATOM 2488 CA PHE 506 41.955 104.935 180.331 1.00 43.64                        ATOM 2500 CA TYR 507 43.306 105.190 183.809 1.00 41.84                        ATOM 2514 CA GLU 508 42.651 101.611 185.218 1.00 39.35                        ATOM 2524 CA THR 509 44.156  99.096 187.597 1.00 41.67                        ATOM 2533 CA PRO 510 46.618  96.563 185.969 1.00 39.70                        ATOM 2540 CA LEU 511 44.134  93.600 186.416 1.00 42.72                        ATOM 2549 CA GLN 512 41.460  95.632 184.624 1.00 37.47                        ATOM 2561 CA LEU 513 43.895  96.148 181.765 1.00 27.02                        ATOM 2570 CA LEU 514 44.769  92.421 181.627 1.00 24.55                        ATOM 2579 CA GLU 515 41.076  91.327 181.688 1.00 31.93                        ATOM 2589 CA LYS 516 40.903  93.442 178.515 1.00 31.96                        ATOM 2602 CA VAL 517 43.981  91.949 176.692 1.00 26.46                        ATOM 2610 CA LYS 518 42.528  88.538 177.852 1.00 30.83                        ATOM 2623 CA ASN 519 39.106  89.397 176.432 1.00 37.62                        ATOM 2634 CA VAL 520 40.221  90.488 172.933 1.00 35.75                        ATOM 2642 CA PHE 521 42.191  87.109 172.662 1.00 25.45                        ATOM 2654 CA ASN 522 39.292  85.079 173.953 1.00 26.10                        ATOM 2665 CA GLU 523 36.870  86.722 171.437 1.00 33.04                        ATOM 2675 CA THR 524 39.260  86.923 168.369 1.00 29.70                        ATOM 2684 CA LYS 525 39.423  83.195 169.011 1.00 28.41                        ATOM 2697 CA ASN 526 35.624  82.663 169.124 1.00 34.46                        ATOM 2708 CA LEU 527 34.816  84.653 166.021 1.00 28.09                        ATOM 2717 CA LEU 528 37.617  82.902 164.068 1.00 25.62                        ATOM 2726 CA ASP 529 35.899  79.662 164.964 1.00 35.00                        ATOM 2735 CA LYS 530 32.659  81.068 163.537 1.00 37.54                        ATOM 2748 CA ASP 531 34.250  82.150 160.272 1.00 37.92                        ATOM 2757 CA TRP 532 38.053  82.453 159.573 1.00 40.02                        ATOM 2773 CA ASN 533 38.395  85.446 157.159 1.00 42.64                        ATOM 2784 CA ILE 534 35.840  87.437 159.393 1.00 39.27                        ATOM 2793 CA PHE 535 38.635  89.996 159.881 1.00 40.49                        ATOM 2805 CA SER 536 38.485  90.720 156.238 1.00 46.56                        ATOM 2813 CA LYS 537 35.844  93.371 157.102 1.00 45.61                        ATOM 2826 CA ASN 538 35.653  96.936 155.760 1.00 54.39                        ATOM 2837 CA CYS 539 35.786  98.626 159.004 1.00 50.55                        ATOM 2844 CA ASN 540 36.738 101.888 157.286 1.00 58.49                        ATOM 2855 CA ALA 541 33.143 102.934 157.648 1.00 61.90                        ATOM 2861 CA SER 542 32.854 101.855 161.326 1.00 56.62                        ATOM 2869 CA PHE 543 35.911 103.888 162.235 1.00 55.25                        ATOM 2881 CA ALA 544 33.885 107.102 161.741 1.00 61.94                        ATOM 2887 CA GLU 545 30.949 107.003 164.203 1.00 63.83                        ATOM 2897 CA CYS 546 33.917 107.418 166.572 1.00 61.90                        ATOM 2904 CA SER 547 33.751 110.878 168.150 1.00 66.22                        ATOM 2912 CA ALA 548 37.461 110.300 168.811 1.00 65.91                      __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AGGTGTCTCA TAGAAAGTTC GGACGCAGGC CTTGTCATGC TCTTCATAAT CC - #TTGG             56                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CAGGAGAAAG CTTATGTCTG AATATTGTAG CGCCATGATT GGGAGTGGAG CC - #CTGCAG           58                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CAGGAGAAAG CTTATGTCTG AATATTGTAG CGCCATGATT GGGAGTGGAC AC - #CTGCAG           58                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CAGGAGAAAG CTTATGTCTG AATATTGTAG CCACATGATT GGGAGTGGAG CC - #CTGCAG           58                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AGGAGAAAGC TTATGTCTGA ATATTGTAGC CACATGATTG GGAGTGGACA CC -             #TGCAGTCT     60                                                                 - - CTGGCTCGGC TG              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GGACGCAGGC CTTGTCATGC TCTTCATAAT CCTTGGTGAA GCAGCTCTTC AG -             #CCTCAAAG     60                                                                 - - AGAGTTCCTG CAGCTGTTTA ATGGC          - #                  - #                   85                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CATGACAAGG CCTGCGTCCG AACTTTCTAT GAGACACCTC TCCAGTTGCT GG -             #CGAAGGTC     60                                                                 - - AAGGCTGTCT TTAATG             - #                  - #                      - #    76                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GGATCAGGAT CCCTCGGACT GCCTCTC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GCGTACCATG GGCCCAGGAG TTCTGC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AGTCGAGGAT CCTCAATCCG GGGGATGCGT GTG       - #                  - #             33                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for identifying candidate humanmacrophage-colony stimulating factor (M-CSF) agonists or antagonists,the method comprising the steps of:a) crystallizing an M-CSF dimer toform at least one M-CSF crystal, the dimer composed of two M-CSFmonomers which may be the same or different, at least one of saidmonomers having at least one but less than five amino acidsubstitutions, the substitutions and residue positions selected from thegroup consisting of His15→Ala or Leu, Gln17→Ala or Glu, Gln79→Ala orAsp, Arg86→Glu or Asp, Glu115→Ala, Glu41→Lys or Arg, Lys93→Ala or Glu,Asp99→Lys or Arg, Leu55→Gln or Asn, Ser18→Ala or Lys, Gln20→Ala or Asp,Arg21→Ala or Glu, Ile75→Lys or Glu, Val78→Lys or Arg, Leu85→Glu or Asn,Asp69→Lys or Arg, Asn70→Ala or Glu, His9→Ala or Asp, Asn63→Lys or Arg,Thr34→Gln or Lys, Cys157→Ser, and Cys159→Ser, one or more of saidmonomers being a full length mature human M-CSF, or being an NΔ3deletion mutein, or having a carboxy truncation ending at a residueposition selected from the group consisting of 158 and 221, or acombination thereof, all said residue positions being relative to maturehuman M-CSF; b) irradiating the M-CSF crystal produced by the procedureof step (a) to obtain a diffraction pattern of the M-CSF crystal; c)determining the three-dimensional structure of M-CSF from thediffraction pattern, the three-dimensional structure including an M-CSFreceptor-binding region; and d) identifying a candidate human M-CSFagonist or antagonist having a three-dimensional structure of the M-CSFbinding region, wherein said candidate M-CSF agonist or antagonist hasan altered signal transduction capacity relative to mature human M-CSFon M-CSF responsive cells.
 2. The method of claim 1 wherein solventaccessible residues do not participate in formation of the M-CSF dimer.3. The method of claim 1, wherein at least one of said M-CSF monomers isselected from the group consisting of His9→Ala M-CSF; His15→Ala M-CSF;NΔ3CΔ158 His9→Ala, His15→Ala M-CSF; Gln20→Ala, Val78→Lys M-CSF; andNΔ3CΔ221 Cys157→Ser, Cys159→Ser M-CSF.
 4. The method of claim 3, whereinat least one of said monomers is His9→Ala M-CSF.
 5. The method of claim3, wherein at least one of said monomers is His15→Ala M-CSF.
 6. Themethod of claim 3, wherein at least one of said monomers is NΔ3CΔ158His9→Ala, His15→Ala M-CSF.
 7. The method of claim 3, wherein at leastone of said monomers is Gln20→Ala, Val78→Lys M-CSF.
 8. The method ofclaim 3, wherein at least one of said monomers is NΔ3CΔ221 Cys157→Ser,Cys159→Ser M-CSF.